WO2011072487A1 - 空间构象改变的重组干扰素的晶体、其三维结构及应用 - Google Patents

空间构象改变的重组干扰素的晶体、其三维结构及应用 Download PDF

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WO2011072487A1
WO2011072487A1 PCT/CN2010/002055 CN2010002055W WO2011072487A1 WO 2011072487 A1 WO2011072487 A1 WO 2011072487A1 CN 2010002055 W CN2010002055 W CN 2010002055W WO 2011072487 A1 WO2011072487 A1 WO 2011072487A1
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woiv
interferon
angstroms
atom
recombinant interferon
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PCT/CN2010/002055
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English (en)
French (fr)
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魏光文
王大成
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四川辉阳生命工程股份有限公司
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Priority to SG2012044889A priority Critical patent/SG181785A1/en
Priority to US13/516,947 priority patent/US8846025B2/en
Priority to CA2784624A priority patent/CA2784624C/en
Priority to AU2010333574A priority patent/AU2010333574B2/en
Priority to CN201080057897.1A priority patent/CN102770447B/zh
Priority to EP10836926.5A priority patent/EP2514761B1/en
Priority to ES10836926.5T priority patent/ES2631028T3/es
Priority to JP2012543445A priority patent/JP2013514279A/ja
Publication of WO2011072487A1 publication Critical patent/WO2011072487A1/zh
Priority to HK13104725.9A priority patent/HK1177749A1/zh
Priority to US14/461,360 priority patent/US9273109B2/en

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Definitions

  • the present invention is primarily directed to crystals of recombinant interferon with altered spatial conformation, methods of crystallization thereof, three-dimensional structures thereof, applications of said crystals and three-dimensional structures, and mimics of said recombinant interferon. Background technique
  • Interferon is a soluble protein produced by a variety of cells that has a variety of important biological functions, including antiviral, antitumor, and immunomodulatory functions. Interferons can be classified into type I, type II, and type III interferons depending on the type of cell produced, the receptor, and the activity of the organism. Type I interferons are primarily induced by viral and synthetic double-stranded RNA and are therefore also referred to as antiviral interferons. There are three types of type I interferons: IFNa, INFp and IFNoo. Type II interferons, also known as immunointerferons or IFNy, are produced by T cells and are an important immunomodulatory factor in vivo. Type III interferons are composed of IFN- ⁇ molecules.
  • U.S. Patent Nos. 4,695,623 and 4,897,471 disclose novel human interferon polypeptides whose amino acid sequences comprise a common or predominant amino acid present in a native a-interferon polypeptide.
  • the novel interferon polypeptide was named IFN-con (common interferon a).
  • the disclosed amino acid sequences were designated as IFN-conl, IFN-con2 and IFN-con3.
  • Genes encoding IFN-cons and gene expression in E. coli are also disclosed. Studies have shown that this recombinant IFN-con has higher antiviral, anti-proliferative and natural killer cell activity in vitro than leukocyte interferon or other type I interferons.
  • U.S. Patent No. 5,372,808 discloses the use of human IFN-con in the treatment of diseases.
  • the recombinant human consensus interferon showed lower side effects than the a-interferon approved by the previous clinical approval, such as the interference energy @ (IFN-a2b, SGP) produced by Schering-Plough.
  • the US Food and Drug Administration FDA approved the interferon produced by Amgen in the United States as a clinical treatment for hepatitis C.
  • the product name is INFERGEN® (interferon alfacon-1). .
  • the crystal quality obtained is not good, the internal structure of the crystal is loose, and the X-ray diffraction resolution is low, reaching only 5 Humans, cannot be applied to further protein spatial structure analysis to obtain useful structural information. Therefore, a crystal of the recombinant interferon having a modified structure and function with good quality and high X-ray diffraction resolution is obtained, the three-dimensional structure thereof is determined, a model is established, and drug design is carried out by using the structure and model. It is very meaningful to improve the efficacy of existing interferons. Summary of invention
  • the present invention relates to a crystal of a recombinant interferon as disclosed in the above-mentioned U.S. Patent No. 7,364,724 and Chinese Patent Publication No. CN1740197A, such a recombinant interferon comprising the amino acid sequence shown in SEQ ID NO: 1. Further, the present invention provides a crystallization method of the above recombinant interferon, a composition comprising the crystal.
  • the present invention provides that the recombinant interferon is different from the three-dimensional structure of interferon such as IFN-oc 2b which has been published in the prior art, and is also different from the three-dimensional structure of the calculation model structure of the American company
  • the three-dimensional structure is used in computer-based identification of candidate compounds that interact with interferons, design of interferon mimics, and rational drug design.
  • the present invention provides a mimetic of the recombinant interferon, a composition comprising the mimetic, and the crystal, mimetic or composition in the preparation of a medicament for treating a viral disease and/or a tumor Applications.
  • Figure 1 shows a single crystal of the recombinant interferon (rSIFN-co) of the present invention for crystal structure analysis.
  • Figure 2 shows the X-ray diffraction pattern of the rSIFN-co crystal (resolution 2.6 A).
  • Figure 3 shows the ⁇ . ⁇ electron density map of the local 2Fo-Fc in the crystal structure of the rSIFN-co molecule.
  • Figure 4 shows a plot of the average temperature factor versus residue for all atoms of rSIFN-co;
  • Figure 4a is a plot of the A chain;
  • Figure 4b is a plot of the B chain.
  • Figure 5 shows the distribution of ( ⁇ , ⁇ ) values of all amino acid residues in the rSIFN-co protein molecular structure model on the Lagrangian conformation map. This figure refers to 118 with a resolution of at least 2.0 angstroms and an R factor of less than 20%. The structure, more than 90% of the best areas are high quality models, the statistics are as follows: Statistical data
  • Residues in the optimal region [A, B, L] 240 90.6%
  • Residues in the additional allowable region [a, b, 1, p] 24 9.1%
  • Residues in the disallowed region 0 0.0% Number of residues other than glycine and proline 265 100.0% Number of terminal residues (except glycine and proline) 127
  • Figure 7 shows the assembled structure of the rSIFN-co dimer structure.
  • Figure 8 shows the organization of the rSIFN-co crystallographic dimer (Fig. 8a, Fig. 8b) and (the root mean square deviation of the carbon atoms (RMSD, the box represents the deleted residue) (Fig. 8c).
  • Figure 9 shows the structure of rSIFN-co single molecule (only the main peptide chain is shown); wherein Figure 9a is a side view; Figure 9b is a top view; Figure 9c is a top view; Figure 9d is a topological structure of the secondary structure .
  • Figure 10 shows a chromatogram of the secondary structure and amino acid sequence of rSIFN-co; wherein the gray line box represents an amino acid residue that is not constructed in the structure, and the blue line box represents an amino acid residue constructed as Ala or Gly in the structure.
  • the connection is a pair of disulfide bonds, and the subscript green is a pair of disulfide bonds that have been constructed in the structure.
  • Figure 11 shows a sequence comparison of the rSIFN-co protein and the homologous IFN polypeptide.
  • Figure 12 shows a comparison of the structural differences between rSIFN- co and IFN-oc2b.
  • Figure 13 shows the overlay of rSIFN-co (red) and IFN-oc2b (yellow) dimer.
  • Figure 14 shows a comparison of the difference between the three-dimensional structure of rSIFN-co and the computational model structure of Infingen.
  • Figure 15 shows (a) the binding model of IFN-oc-like protein to the receptor; (b) Schematic diagram of the IFN-o-like functional region (blue circle is an important functional region).
  • Figure 16 shows the mean blood concentration-time curve of 18 subjects after subcutaneous injection of rSIFN-co 9 ⁇ g and INFERGEN® 9 ⁇ g. Detailed description of the invention
  • the purified product of the recombinant interferon of the present invention before crystallization is obtained by the method described in the descriptions of Examples 1 and 2 of the specification of U.S. Patent No. 7,364,724 and/or on the pages 11 to 17 of the specification of Chinese Patent Publication No. CN1740197A.
  • the definition of the recombinant interferon of the present invention reference is made to U.S. Patent No. 7,364,724 and/or Chinese Patent Publication No. CN1740197A.
  • the amino acid sequence of the recombinant interferon of the present invention and the nucleotide sequence encoding the amino acid are as follows:
  • the circular dichroism of the recombinant interferon of the present invention in the range of 190-250 nm is significantly different from the circular dichroism of dry hydrazine measured under the same conditions, at 250-320 nm.
  • the circular dichroism in the range is also significantly different from the circular dichroism of dry hydrazine (see Chinese Patent Publication No. CN1740197A, page 3, lines 22-25, Example 3, and Figs. 6A-D).
  • the three-dimensional structure of the recombinant interferon of the present invention is different from the three-dimensional structure of the IFN-cc 2b which has been published in the prior art (see Fig. 12), and is also different from the computational model structure of the dry complex (see KORN, AP, etc., Journal of Interferon Research 1994, 14: 1-9), the results of the comparison between the two show that the AB ring of the two is clearly different, and the BC ring can not completely overlap (see Figure 14).
  • the recombinant interferon of the present invention is administered intramuscularly to a human body having a body mass index (BMI) of 18 to 23, and the blood sampling time is 2-5
  • BMI body mass index
  • a oligonucleotide enzyme in the serum of the test subject also referred to as For the concentration of "2', 5'-OAS"
  • the curve obtained generally shows a double peak, and the area under the curve is significantly larger than the area under the curve obtained by injecting dry hydrazine under the same conditions.
  • the recombinant interferon of the present invention has a half-life in the human body after injection into the human body, half of that in the human body. The decay is longer.
  • the recombinant interferon of the present invention has a stronger potency than any other interferon currently used in clinical use (including dry hydrazine).
  • the recombinant interferon of the present invention can inhibit not only the DNA replication of the hepatitis B virus but also the secretion of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg), and inhibit the hepatitis B virus core.
  • the efficacy of antigen (HBcAg) DNA replication is approximately double that of that of Qianfujin.
  • the in vitro pharmacodynamics of the recombinant interferon of the present invention shows that it can inhibit not only the DNA replication of hepatitis B virus, but also the secretion of surface antigen and e antigen, and its cytotoxicity is only 1/8 of that of clinically active interferon.
  • the antiviral activity is 5-20 times that of the clinically active interferon, and at the same time, it has a higher and broader biological response in the human body.
  • the interferon has higher antiviral activity and lower side effects than other interferons (including dry hydrazine), both for the prevention of viral diseases and for the treatment of tumors.
  • the recombinant interferon of the present invention not only has 20 times more antiviral activity than clinically active interferon, but also is significantly stronger than recombinant human alpha interferon (including dried hydrazine), such as anti-tumor and cervical cancer. It also greatly reduces the side effects and can be safely used in large doses (> 10 million IU per dose), making it possible to successfully treat some viral diseases or tumors requiring high doses of interferon.
  • the recombinant interferon used in the present invention has a different spatial conformation, enhanced biological activity, and different pharmacokinetic properties than dry hydrazine.
  • spatial conformation As used herein, the terms “spatial conformation”, “space structure”, “three-dimensional structure”, and “three-dimensional conformation” are used interchangeably.
  • the recombinant interferon used in the present invention comprises the amino acid sequence set forth in SEQ ID NO: 1, and is encoded by the nucleotide sequence comprising SEQ ID NO: 2. Further, the amino acid sequence of the recombinant interferon used in the present invention is shown by SEQ ID NO: 1, and is encoded by the nucleotide sequence shown in SEQ ID NO: 2. Further, the recombinant interferon is interferon encoded with an amino acid sequence as shown in SEQ ID NO: 1 (or with the same amino acid sequence) but not as the nucleotide sequence shown in SEQ ID NO: 2.
  • the recombinant interferon has altered spatial conformation and enhanced biology. Active, altered spatial conformation And different pharmacokinetic properties, or with enhanced biological activity and different pharmacokinetic properties.
  • the altered spatial conformation comprises: the primary dichroism of the recombinant interferon in the range of 190-250 nm is significantly different from the circular dichroism of the thiophanate determined under the same conditions and/or at 250-320 nm
  • the circular dichroism in the range is also significantly different from the circular dichroism of dry hydrazine
  • the enhanced biological activity includes: enhanced antiviral activity, enhanced antitumor activity, lower side effects and/or high dose use.
  • the dosage may be > 10 million IU per dose.
  • the enhanced biological activity may be enhanced antiviral activity and enhanced antitumor activity, etc.
  • the tumor may be breast cancer and cervical cancer.
  • the different pharmacokinetic properties include: After intramuscular injection to a human body with a body mass index of 18-23, the blood sampling time is used to determine the concentration of 2-5 A oligonucleotide in the serum of the test subject. In the graph, the area under the curve of the resulting curve is significantly larger than the area under the curve obtained by injecting dry hydrazine under the same conditions and/or a longer half-life compared to hydrazine.
  • the recombinant interferon used in the present invention can be produced by a method comprising: introducing a nucleotide sequence comprising SEQ ID NO: 2 encoding a recombinant interferon into an isolated host cell, The host cell is cultured under conditions suitable for expression of the recombinant interferon, and the recombinant interferon is harvested, wherein the recombinant interferon has the amino acid sequence of SEQ ID NO: 1, and the recombinant interferon inhibits B Hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) secretion.
  • the host is Escherichia coli, such as. E. coli LGM 194.
  • the nucleotide sequence comprising SEQ ID NO: 2 is under the control of the promoter P ⁇ D .
  • the harvesting step comprises extracting interferon from the culture fermentation broth, collecting inclusion bodies, denaturation and renaturation of the harvested interferon. Still further, the harvesting step further comprises isolating and purifying the recombinant interferon (see U.S. Patent No. 7,364,724). Recombinant interferon crystal and crystallization method
  • the present invention provides a crystal of the recombinant interferon of the present invention.
  • the metal ions may be magnesium ions, zinc ions or the like, and these metal ions may mediate the formation of interferon dimers in the crystal.
  • the recombinant interferon is encoded by a nucleotide sequence comprising SEQ ID NO: 2.
  • the present invention provides a crystal of a recombinant interferon comprising the amino acid sequence of SEQ ID NO: 1, preferably having an amino acid sequence as shown in SEQ ID NO: 1, wherein the crystal
  • such a recombinant interferon is encoded by a nucleotide sequence comprising SEQ ID NO: 2, preferably by a nucleotide sequence as set forth in SEQ ID NO: 2.
  • the present invention provides a method of preparing (cultivating) a recombinant interferon crystal of the present invention.
  • the present invention provides a method of preparing (cultivating) a recombinant interferon crystal of the present invention, comprising the steps of: concentrating recombinant interferon to about 3-3.5 mg/ml, and placing Li 2 S0 4 , CAPS
  • the crystal is obtained under a crystallization solution condition of (3-(cyclohexylamino)-1-propanesulfonic acid) and MgCl 2 for a suitable period of time. Further, the crystal growth method is carried out at room temperature such as 293K. In certain embodiments, the crystal culture is carried out by a hanging drop method or a drop method, preferably by a hanging drop method (also referred to as a hanging drop vapor phase diffusion method). In certain embodiments, the crystallization solution conditions comprise from about 1.0 to about 1.5 M Li 2 S0 4 , from about 0.05 to about 0.15 M CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), and from about 0.01 to about 0.03 M MgCl 2 . In certain embodiments, the crystallization solution conditions have a pH in the range of from about 10.5 to about 12.0, preferably about 11.1. In certain embodiments, the crystallization solution conditions are 1.2 M Li 2 S0 4 , 0.1 M CAPS
  • the crystal culture comprises allowing the above-described recombinant interferon-containing crystallization solution to stand for from about 1 day to about 2 weeks, preferably from about 2 days to about 10 days, more preferably from about 3 days to about 1 week. For example, 3 days - 1 week.
  • X-ray crystallography analysis Each of the constituent amino acids of the interferon disclosed herein is defined by a set of structural coordinates (also referred to as "atomic coordinates").
  • structural coordinate refers to a Cartesian coordinate derived from a mathematical equation relating to a pattern obtained by X-ray monochromatic light obtained by atomic (scattering center) diffraction of the interferon of the present invention in the form of crystal.
  • the diffraction data is used to calculate the electron density map of the crystal repeat unit.
  • the electron density map is then used to establish the position of a single atom of the interferon protein or protein/ligand complex.
  • a slight structural coordinate change can also be produced by mathematically operating the interferon or interferon/ligand structure coordinates.
  • the structural coordinates disclosed herein can be manipulated by: crystallographic permutation of structural coordinates, segmentation of structural coordinates, overall addition and subtraction of a set of structural coordinates, flipping of structural coordinates, or any combination of the above. Changes in crystal structure due to amino acid mutations, additions, substitutions and/or deletions or other changes in any of the constituents can also cause changes in structural coordinates. Such slight changes in individual coordinates have little effect on the overall shape. If such a change is within acceptable standard error compared to the original coordinates, the resulting three-dimensional shape is considered structurally equivalent.
  • the "AB loop" of the recombinant interferon of the present invention refers to the amino acid residue at positions 25-33 of the recombinant interferon of the present invention (the amino acid sequence of which is represented by SEQ ID NO: 1), that is, the AB loop.
  • the sequence of the amino acid sequence SPFSCLKDR shown in SEQ ID NO: 4; the "BC loop” of the recombinant interferon of the present invention refers to amino acids 44-52 of the recombinant interferon of the present invention (the amino acid sequence of which is shown in SEQ ID NO: 1).
  • the residue, that is, the sequence of the BC loop is the amino acid sequence DGNQFQKAQ shown by SEQ ID NO: 5.
  • the phrase "in combination with” refers to the condition in which a ligand or a portion thereof is adjacent to an interferon molecule or a portion thereof.
  • Such a combination may be non-covalent, wherein such proximity is energetically beneficial to hydrogen bonding, van der Waals forces or electrostatic interactions; the binding may also be covalent.
  • a ligand that binds to a binding pocket or binding region of an interferon should also be expected to bind to, or interact with, a structurally equivalent binding pocket or binding region.
  • any molecular or molecular complex or any portion thereof, when superimposed to the relevant backbone atom described herein, has a conserved residue backbone atom of less than about 0.65 angstroms (eg, N, Ca, C, 0, preferably Ca) Root mean square deviation is considered to be "structurally equivalent". In other words, the crystal structures of these sites of the two molecules are substantially identical within an acceptable tolerance.
  • Particularly preferred structurally equivalent molecules or molecular complexes are molecules or molecular complexes as defined below: structures disclosed herein The complete set of coordinates ⁇ 'j, the root mean square deviation from the conservative backbone atoms of those amino acids at about 0.65 angstroms.
  • the root mean square deviation is at most about 0.5 angstroms, still more preferably at most about 0.35 angstroms.
  • Other embodiments of the invention include molecular complexes having the following: The structural coordinates of the AB or BC ring disclosed herein are less than a root mean square deviation of about 0.65 Angstroms, preferably the root mean square deviation is at most about 0.5 Angstroms, More preferably, it is at most about 0.35 angstroms.
  • root mean square deviation refers to the square root of the arithmetic mean of the square of the deviation used to indicate deviations or changes from trends or objects.
  • the "root mean square deviation” defines a change in a protein backbone relative to an interferon backbone or portion thereof as defined by the structural coordinates as described herein.
  • the X-ray structure coordinates define the unique conformation of points in space.
  • the structural coordinates of the protein, or protein/ligand complex, or a portion thereof define a series of corresponding points; these points in turn define a three-dimensional conformation. If the distance and angle between the coordinates remain substantially the same, a similar or identical conformation can be defined for a set of completely different coordinates.
  • the scalable point constellation can be defined by increasing or decreasing the distance between the coordinates.
  • Various computational analyses can be used to determine whether a molecule or a portion thereof and the interferon disclosed herein or a portion thereof are "structurally equivalent" as defined by the three-dimensional structure. For example, comparisons between different structures, different conformations of the same structure, or different portions of the same structure can be performed by various computational analyses.
  • the analysis can include the following four steps: (1) loading the structure to be compared; (2) defining atomic equivalence in these structures; (3) performing a fitting operation; and (4) Analysis results.
  • the present invention provides a three-dimensional structure of the recombinant interferon of the present invention.
  • This three-dimensional structure is different from the three-dimensional structure of IFN-a2b that has been published in the prior art (see Fig. 12), and is also different from the computational model structure of Amgen's dry complex (see Fig. 14), especially in the AB ring and There are structural differences between BC ring and IFN-a 2b and dry hydrazine.
  • the three-dimensional structure of the recombinant interferon comprises the atomic coordinates of the recombinant interferon as shown in Table 7, optionally at less than about 0.65 Angstroms, preferably about 0.5 Angstroms and more preferably about 0.35.
  • the conserved residue backbone atom of angstrom preferably changes within the root mean square deviation of the Ca atom (also referred to as "a carbon atom").
  • each of the recombinant interferons Each monomer consists of a 6-segment alpha helix, a 1 segment 3 10 helix, and a linker peptide therebetween.
  • the amino acid residue positions corresponding to the 6-segment alpha helix are 13-20, 50-68, 70-76, 79-100, 114-133, and 138-160, respectively; the 1 segment 3 1() helix corresponds to The amino acid residue position is 40-43.
  • the folding structure of the monomer structure belongs to a helical cytokine type, characterized in that: the alpha carbon atom skeleton of the recombinant interferon and the carbon atom skeleton of the IFN-a2b protein are superposed by a least squares method. Thereafter, the position of each of the carbon atoms of the recombinant interferon at positions 25-33 (AB ring) and the carbon atoms of the IFN-a2b protein at the corresponding residue are both deviated (location root -mean-square deviation ) is 3.63 angstroms ⁇ 5%.
  • the positional root mean square deviation of the alpha carbon atom of the recombinant interferon from the IFN-a2b protein at residue 25 is 3.291 angstroms ⁇ 5%; the alpha carbon at the 26th residue
  • the positional root mean square deviation of the atom is 4.779 angstroms ⁇ 5%; the positional root mean square deviation of the alpha carbon atom at the 27th residue is 5.090 angstroms ⁇ 5%; the alpha carbon atom at the 28th residue
  • the positional root mean square deviation is 3.588 angstroms ⁇ 5%;
  • the position of the a carbon atom at the 29th residue has a root mean square deviation of 2.567 angstroms ⁇ 5%;
  • the position of the alpha carbon atom at the 30th residue is The square root deviation is 2.437 ⁇ ⁇ 5%; the positional root mean square deviation of the ⁇ carbon atom at the 31st residue is 3.526 ⁇ ⁇ 5%; the position rms of
  • each of the recombinant interferon at positions 44-52 (BC ring) (the position of the X carbon atom and each ⁇ carbon atom of the IFN-a2b protein at the corresponding residue)
  • the root mean square deviation is 2.90 angstroms ⁇ 5%.
  • the positional root mean square deviation of the carbon atom at the 44th residue of the recombinant interferon and the IFN-oc2b protein is 1.614 angstroms ⁇ 5%, in the first
  • the positional root mean square deviation of the oc carbon atom at position 45 is 1.383 angstroms ⁇ 5%, and the position of the carbon atom at the 46th residue has a root mean square deviation of 2.735 angstroms ⁇ 5%.
  • the positional root mean square deviation of the oc carbon atom on the base is 2.709 angstroms ⁇ 5%, and the positional root mean square deviation of the alpha carbon atom at the 48th residue is 5.018 angstroms ⁇ 5%, at the 49th residue.
  • the positional root mean square deviation of the alpha carbon atom is 4.140 angstroms ⁇ 5%, and the positional root mean square deviation of the a carbon atom at the 50th residue is 3.809 angstroms ⁇ 5%, and the alpha at the 51st residue
  • the positional root mean square deviation of the carbon atom is 2.970 angstroms ⁇ 5%, and the positional root mean square deviation of the alpha carbon atom at the 52nd residue is 0.881 angstroms ⁇ 5% (the position listed above)
  • Root bias "are the coordinates of the location of the root mean square deviation).
  • the invention provides a portion of the selected three-dimensional structure of a recombinant interferon of the invention, wherein the selected portion of the three-dimensional structure comprises amino acids 25-33 as contained in Table 7.
  • "one or more amino acid residues" as described herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 amino acid residues.
  • “the selected portion of the three-dimensional structure” comprises the atomic coordinates from amino acid residues 25-33 and/or amino acid residues 44-52 contained in Table 7.
  • the selected portion of the three-dimensional structure comprises at least 5, 6, 7, 8, 9, 10, 11, as contained in Table 7.
  • the above atomic coordinates are optionally varied within a root mean square deviation of a conserved residue backbone atom, preferably a Ca atom, of less than about 0.65 Angstroms, preferably about 0.5 Angstroms, and more preferably about 0.35 Angstroms.
  • the invention provides a proteinaceous spatial structure model comprising a three dimensional structure of a recombinant interferon of the invention.
  • the protein spatial structure model may be an electron density map, a wireframe model, a mesh model, a space fill model, a stick model, a ribbon model, a molecular surface model, and the like.
  • the present invention provides a scalable three-dimensional conformation consisting of dots, at least a portion of which are derived from the structural coordinates disclosed herein, or derived from a recombinant interferon comprising the present invention.
  • the scalable three-dimensional conformation consisting of dots is displayed as a hologram, a stereogram, a model, or a computer display image.
  • the invention provides a method of screening/designing a candidate compound that interacts with a recombinant interferon of the invention. Further, the method utilizes the three-dimensional structure of the recombinant interferon of the present invention, and further, the method is computer based. In one embodiment, the invention provides a computer-based method of identifying candidate compounds capable of interacting with recombinant interferon, the method comprising (a) providing a three-dimensional structure comprising atomic coordinates of the recombinant interferon shown in Table 7, optionally at a conserved residue backbone of less than about 0.65 Angstroms, preferably about 0.5 Angstroms and more preferably about 0.35 Angstroms.
  • the atom preferably changes within the root mean square deviation of the Ca atom; and (b) selects a candidate compound having a structural feature capable of interacting with the above-described three-dimensional structure or a selected portion of the three-dimensional structure, thereby determining the ability to interact with the recombinant interferon Candidate compounds for interaction.
  • the structural feature is selected from the group consisting of an antigenic site, a hydrophilic property, a surface accessibility, and a structural motif.
  • the selection and identification of candidate compounds in step (b) comprises: (i) generating a three-dimensional structure for a plurality of candidate compounds; and (ii) each of the three-dimensional structures of step (i) and the steps described above) The three-dimensional structure in or the selected portion of the three-dimensional structure is fitted to find the most energy-efficient interaction to identify candidate compounds that interact with the recombinant interferon.
  • the method further comprises the steps of: (c) acquiring or synthesizing a candidate compound; and (d) contacting the candidate compound with the recombinant interferon to determine the candidate compound and the recombinant interferon The ability to interact.
  • the step of determining the ability of the candidate compound to interact with the recombinant interferon can further comprise determining the activity of the recombinant interferon upon contact with the candidate compound.
  • the interferon activity to be tested includes: antiviral activity, antitumor activity, antiproliferative activity, natural killer cell activity, and immunomodulatory activity.
  • the candidate compound is a ligand that binds to the recombinant interferon or a selected portion thereof.
  • the ligand is selected from the group consisting of: a receptor, a modifier, an agonist, and an antagonist.
  • such a receptor can be IFNAR1, IFNAR2, or a complex thereof; and wherein the selected Partially comprising one or more amino acid residues of amino acid residues 25-33 of the recombinant interferon (sequence of the AB loop) and/or amino acid residues 44-52 (sequence of the BC loop). Further, wherein the selected portion comprises amino acid residues 25-33 and/or amino acid residues 44-52 from the recombinant interferon.
  • the invention provides a method of identifying a potential ligand that binds to a recombinant interferon of the invention.
  • the method comprises the steps of: exposing the crystals disclosed herein to one or more samples comprising a potential ligand and determining whether a ligand-interferon molecular complex is formed.
  • the invention provides a method of obtaining structural information for designing a potential ligand capable of forming a molecular complex with an interferon.
  • the method comprises the steps of: exposing the crystals disclosed herein to a library of potential ligands and determining whether a ligand-interferon molecule complex is formed.
  • the invention provides a computer-aided identification, design or preparation of a potential dry A method of interferon activity modulator.
  • the method comprises screening a chemical or biological entity library.
  • a preferred method can include the steps of: obtaining unliganded interferon crystals; exposing the unliganded interferon crystals to one or more tests comprising potential interferon ligands a sample; and determining whether a ligand-interferon molecular complex is formed.
  • the interferon can be exposed to a potential ligand by a variety of methods including, but not limited to, immersing the interferon crystal in a solution containing one or more potential ligands, or, in one or more Co-crystallized interferon in the presence of a potential ligand.
  • Structural information from the ligand-interferon complex can preferably be used to design new ligands that bind more tightly, bind more specifically than known ligands, have desirable biological activity characteristics, Higher security, even with the combination of the above advantages.
  • the calculated electron density map directly reveals binding events, identifies bound chemical or biological entities, and provides a detailed three-dimensional structure of the ligand-interferon complex.
  • a series of analogs or derivatives of the hit with more tightly bound or desired biological activity can be identified by conventional screening methods.
  • the ligand-interferon can be iteratively exposed to additional potential ligands such that two or more hits can preferably be combined together to identify or design a more potent ligand.
  • the structural coordinates disclosed herein can be used to help obtain structural information about other crystalline molecules or molecular complexes.
  • the methods of the present invention can be used to determine at least a portion of the three-dimensional structure of a molecule or molecular complex having one or more similar structural features to the stem disclosed herein.
  • the molecules are referred to herein as "structurally homologous.” Similar structural features may include, for example, regions of amino acid identity, conserved active sites or binding site motifs, and similarly arranged secondary structural elements (eg, alpha helices and beta sheets).
  • structural homology is determined by aligning residues of two amino acid sequences to optimize the number of identical amino acids in their sequence length; in performing the alignment, allowing for further Vacancies exist in one or both to optimize the number of identical amino acids, however, these amino acids must maintain an inherent sequence in their respective sequences.
  • the structurally homologous molecule is a protein having an amino acid sequence at least 65% identical to SEQ ID NO: 1. More preferably, the protein homologous to the interferon structure of the invention comprises at least 80% amino acid sequence identity to a similar portion of SEQ ID NO: 1. A continuous segment of less than 50 amino acid residues.
  • the structural coordinate system disclosed herein can also be used to resolve the crystal structure of interferon homologs, interferon mutants, or interferon homologs co-combined with various ligands.
  • This application makes it possible to determine the optimal site for interaction between ligands and interferons, such as candidate interferon modifiers and interferons.
  • the method is also suitable for identifying potential sites for modification in a plurality of binding sites of a molecule. This information is obtained by providing an additional pathway for determining the most effective binding interactions, such as enhanced hydrophobic interactions between interferons and their ligands.
  • the present invention also provides a method for recombining an interferon mimetic based on a computer design, comprising the steps of: (a) generating a three-dimensional structure for a plurality of mimics; and (b) step (a) The three-dimensional structures of the plurality of mimics are each fitted to a three-dimensional structure containing the atomic coordinates of the recombinant interferon shown in Table 7 or a selected portion of the three-dimensional structure to find a best fit simulation of the recombinant interferon
  • the atomic coordinates are optionally varied within a root mean square deviation of a conserved residue backbone atom, preferably a Ca atom, of less than about 0.65 angstroms, preferably about 0.5 angstroms, and more preferably about 0.35 angstroms.
  • Computer technology can be used to screen, identify, select, and/or design chemical entities or ligands that bind to interferons or structurally homologous molecules.
  • the information on the structural coordinates of the interferons disclosed herein makes it possible to design and/or identify synthetic compounds and/or other molecules having complementary conformations to the conformation of the interferon disclosed herein.
  • computer technology can be used to identify or design chemical entities or ligands, such as receptors, modifiers, agonists, and antagonists, that bind to interferons or portions thereof, such as the AB or BC loop.
  • a potential modifier may bind to, or interfere with, the active site or portion of the interferon, and may be a competitive, non-competitive or non-competitive inhibitor; or by combining between two monomers The interface thus interferes with dimerization.
  • these inhibitors/agonists/antagonists can be used therapeutically or prophylactically to block or enhance interferon activity.
  • the structure-activity data of the interferon binding and interfering ligand analogs can also be obtained by computer technology.
  • chemical entity refers to a compound, a complex of two or more compounds, and a fragment of the compound or complex.
  • Chemical entities that are determined to bind to the interferon of the present invention are potential drug candidates.
  • a three-dimensional structural representation of the results of an interferon or structural homologous molecule or portion thereof of the invention as identified herein can be advantageously used in drug discovery.
  • the structural coordinate system of the chemical entity can be used to generate three dimensions by one of a variety of computer methods and techniques available in the art. An image, which in turn can be fitted to the three-dimensional image of the interferon or structural homologous molecule by a computer.
  • the drug design method of the invention comprises assessing the potential binding of a known chemical entity or ligand to the interferon or its structural homologous molecule. Accordingly, the method of drug design further includes evaluating, by a computer, the potential binding of the selected chemical entity or ligand to any one of the molecular or molecular complexes listed herein.
  • the pharmaceutical design method comprises a computer aided design of a chemical entity or ligand that binds to an interferon of the invention, a homolog thereof, or a portion thereof. Chemical entities or ligands can be designed in a step-by-step fashion, either in one piece at a time, or as a whole or de novo, design.
  • the present invention provides a computer-based rational drug design method comprising the steps of: (a) providing a three-dimensional structure comprising atomic coordinates of the recombinant interferon shown in Table 7, said atomic coordinates Optionally, within a root mean square deviation of a conserved residue backbone atom of less than about 0.65 Angstrom, preferably about 0.5 Angstrom, and more preferably about 0.35 Angstrom, preferably a Cot atom; (b) providing a plurality of molecular fragments and generating a three-dimensional structure thereon (c) fitting each of the three-dimensional structures of the plurality of molecular fragments to the three-dimensional structure of step (a) or the selected portion of the three-dimensional structure; and (d) assembling the selected molecular fragments into one The molecule is used to produce a candidate drug.
  • the method further comprises the steps of: (e) obtaining or synthesizing a candidate drug; and (f) contacting the candidate drug with the recombinant interferon to determine that the candidate drug and the recombinant interferon are mutually The ability to function.
  • the selected portion of the three-dimensional structure comprises amino acid residues from position I 25-33 in Table 7 (amino acid sequence set forth in SEQ ID NO: 4) and/or 44- The atomic coordinates of one or more amino acid residues of the amino acid residue at position 52 (amino acid sequence of SEQ ID NO: 5), and further, the selected portion of the three-dimensional structure comprises from pages 25-33 in Table 7.
  • a conserved residue skeleton atom of about 0.5 angstrom and more preferably about 0.35 angstrom preferably has a variation in the root mean square deviation of the Ca atom.
  • a computer model of an uncrystallized interferon homolog can be constructed or optimized by homology modeling.
  • Calculation software can be used to implement the sequence alignment and secondary structure predictions described.
  • Structural inconsistencies such as the insertion or absence of structural fragments formed around can be obtained by screening a library of peptide structures of the desired length and suitable conformation.
  • the final homology model can be used to resolve the crystal structure of the homologue by techniques known in the art.
  • the energy minimization model can include regions that are subject to stereochemical constraints, in which case the regions can be remodeled using techniques known in the art to obtain the final homology model.
  • the invention provides a method of obtaining structural information about an unknown structural molecule or molecular complex.
  • the method comprises the steps of: crystallizing the molecule or molecular complex; generating an X-ray diffraction pattern from the crystallized molecule or molecular complex; and applying the X-ray diffraction pattern to The structural coordinates of at least a portion of the disclosed interferon, thereby producing a three-dimensional electron density map of at least a portion of the unknown structural molecule or molecular complex.
  • the invention provides a method of modeling an interferon homolog.
  • the method comprises the steps of: aligning the amino acid sequence of the putative interferon homolog with the amino acid sequence of the recombinant interferon of the invention and incorporating the amino acid sequence of the putative interferon homologue In the interferon model formed by the structural coordinates disclosed herein, thereby generating a primary model of the interferon homolog; performing energy minimization on the primary model to generate an energy minimization model; reconstructing the energy minimization model in violation of stereochemistry The constrained region, thereby generating the final model of the interferon homolog.
  • the present invention provides interferon mimetics.
  • the invention provides a peptide comprising a sequence disclosed herein, or a derivative, active moiety, analog, variant or mimetic thereof, and uses thereof. Accordingly, in one embodiment, the invention provides an interferon mimetic comprising the amino acid sequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 5.
  • the interferon mimetic after interfering with the alpha carbon atom skeleton in the three-dimensional structure of the interferon mimetic and the alpha carbon atom skeleton in the three-dimensional structure of IFN-a2b in a least squares manner, corresponds
  • the a-carbon root deviation of each a carbon atom at positions 25-33 in the IFNa2b protein molecule and the IFNa2b protein molecule at the corresponding residue is 3.63 angstroms ⁇ 5%.
  • the interferon mimetic a carbon atom at residues 25-33 is compared to the corresponding residue at IFNa2b
  • the deviations are 3.291 angstroms ⁇ 5%, 4.779 angstroms ⁇ 5%, 5.090 angstroms ⁇ 5%, 3.588 angstroms ⁇ 5%, 2.567 angstroms ⁇ 5%, 2.437 angstroms ⁇ 5%, 3.526 angstroms ⁇ 5%, 4.820 angstroms ⁇ 5% And 2.756 angstroms ⁇ 5%.
  • the interferon mimetic is The root mean square deviation of a carbon atom corresponding to the a-carbon atom at positions 44-52 in the IFNa2b protein molecule and the IFNa2b protein molecule at the corresponding residue is 2.90 angstroms ⁇ 5%.
  • the a carbon atom deviation of the interferon mimetic at residues 44-52 is 1.614 angstroms ⁇ 5%, 1.383 angstroms ⁇ 5%, respectively, compared to the corresponding residue of IFNa2b. 2.735 angstroms ⁇ 5%, 2.709 angstroms ⁇ 5%, 5.018 angstroms ⁇ 5%, 4.140 angstroms ⁇ 5%, 3.809 angstroms ⁇ 5%, 2.970 angstroms ⁇ 5%, and 0.881 angstroms ⁇ 5%.
  • the interferon mimetic is a functional mimetic or a structural mimetic.
  • the interferon mimetic is a mimetic of a recombinant interferon (rSIFN-co) of the invention, and further, the interferon mimetic does not comprise dry hydrazine.
  • the three-dimensional structure of the interferon mimetic is similar to the three-dimensional structure of the recombinant interferon (rSIFN-co) of the invention, particularly at the positions of the AB and BC loops corresponding to the recombinant interferon of the invention, The three-dimensional structure of the two can be substantially the same or identical.
  • the three-dimensional structure of the interferon mimetic comprises atomic coordinates of amino acid residues 25 to 33 (AB ring) and/or amino acid residues 44 to 52 (BC ring) in Table 7,
  • the atomic coordinates are optionally varied within a root mean square deviation of the conserved residue backbone atoms, preferably Ca atoms, of less than about 0.65 Angstroms, preferably about 0.5 Angstroms, and more preferably about 0.35 Angstroms.
  • the invention encompasses variant peptides in which individual amino acids can be substituted with other amino acids that are considered to be closely related in the art.
  • the individual amino acid can be substituted by: replacing any other hydrophobic aliphatic amino acid with any hydrophobic aliphatic amino acid; and replacing any other hydrophobic aromatic amino acid with any hydrophobic aromatic amino acid; Neutral amino acids in polar side chains replace other neutral amino acids with polar side chains; other acidic amino acids can be substituted with any acidic amino acid; other basic amino acids can be substituted with any basic amino acid.
  • mimetic refers to a peptide variant or organic compound having the same functional/structural activity as the polypeptides disclosed herein.
  • Examples of such mimetics or the like include: a three-dimensional structure modeled to interferon disclosed herein (the three-dimensional structure comprising the atomic coordinates of the recombinant interferon shown in Table 7), particularly as described above A compound or peptide of an arrangement of amino acid residues.
  • the mimetic of recombinant interferon of the invention refers to having the same function as the recombinant interferon (rSIFN-co) of the invention/ A peptide variant or an organic compound which is structurally active but different from the recombinant interferon of the present invention, especially a peptide having the same spatial structure as the AB loop and/or BC loop of the recombinant interferon of the present invention, but different from the recombinant interferon of the present invention. Body or organic compound.
  • the amino acid sequence of such a "mimetic" generally has a length similar to the amino acid sequence of the recombinant interferon of the present invention, for example, may comprise from about 120 to 200 amino acid residues, preferably from about 140 to 180 Amino acid residue, more preferably about 150-175 amino acid residues, still more preferably about 160-170 amino acid residues, such as about 164, 165, 166 or 167 amino acid residues; or, such a "mimetic” can Is a peptide variant having a shorter amino acid sequence length of the AB loop and/or BC loop comprising the recombinant interferon of the invention, for example it may comprise from about 10 to 100 amino acid residues, preferably from about 15 to 80 amino acid residues.
  • Suitable mimetics or the like can be generated by molding techniques generally known in the art. This includes designing "mimetics” that involve studying functional interactions and designing compounds that contain functional groups arranged in such a way as to produce the above interactions.
  • Designing mimetics for known pharmaceutically active compounds is a known method for developing drugs based on "leading" compounds. This method can be used to obtain an active compound which is difficult to synthesize or requires high-priced synthesis, and can also be used to improve an active compound which is not suitable for conventional administration; the active compound which is not suitable for conventional administration includes: rapid decomposition by a protease in the digestive tract, Active compound polypeptides which are not suitable for oral administration. Mock design, synthesis, and testing can be used to avoid random screening of large numbers of molecules for target properties.
  • a specific portion of a compound/peptide that plays a key and/or important role in determining the target property is determined.
  • determination can be achieved by systematically altering the amino acid residues in the peptide, for example by substituting each residue in turn.
  • moieties or residues constituting the active region of the compound are referred to as "pharmacophores”.
  • the pharmacophore Once the pharmacophore is found, its structure can be modeled according to its physical properties; such physical properties as stereochemistry, bonding, size and/or charge, which are used from a variety of sources such as spectroscopy Technical, X-ray diffraction and NMR data. Computational analysis, similarity mapping (which simulates the charge and/or volume of the pharmacophore, rather than interatomic bonding) and other techniques can be used in the modeling process. In a variation of the method, the three-dimensional structure of the ligand and its binding partner is modeled. This method is particularly useful where the ligand and/or binding partner produces a conformational change in the binding, allowing for further consideration of the model in the mock design.
  • a template molecule to which a chemical group that mimics the pharmacophore is transplanted is selected.
  • the template molecule and the chemical group grafted thereto can be conveniently selected to readily synthesize the mimetic; and the synthesized mimetic may be pharmacologically acceptable in addition to maintaining the biological activity of the lead compound And does not degrade in the body.
  • the mimetic obtained by this means is screened, it can be known whether it has a target property or to what extent the target property is displayed. Further optimization and modification can then be performed to obtain one or more final mimetics for in vivo or clinical testing.
  • the invention also provides an unligandized molecule; said molecule having at least a portion of an interferon disclosed herein.
  • the non-liganding-binding molecule may comprise the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 (respectively the AB loop of the interferon disclosed herein and the sequence ⁇ 'J of the BC loop, respectively), further
  • the non-liganded molecule has the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • compositions comprising recombinant interferon crystals of the invention or recombinant interferon mimetics of the invention.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the preferred dosage is "prophylactically effective dose” or "treatment”
  • the effective dose is such a dose sufficient to show beneficial effects on the individual.
  • the actual dosage, frequency of administration, and time of administration of the present invention will depend on the nature and severity of the condition being treated. Treatment prescriptions, such as dose determination, are subject to availability by the doctor or other health care provider.
  • the pharmaceutical composition may be administered alone or in combination depending on the circumstances.
  • compositions for use in accordance with the invention and for use in the present invention may comprise, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known in the art. Such materials should be non-toxic and do not affect the efficacy of the active ingredient.
  • the exact nature of the carrier or other substance depends on the mode of administration; the mode of administration includes oral and injection; and the injection further includes subcutaneous, intramuscular, and intravenous injections. Examples of the above techniques and dosing regimens can be found in Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. (ed.), 1980.
  • the pharmaceutical composition can be formulated into the following dosage forms, including: tablets, capsules, Oral liquid, patch, injection, spray, suppository, solution preparation, the recommended dosage form is injection. It can be administered subcutaneously or intravenously.
  • the carrier in the pharmaceutical composition can be any suitable acceptable pharmaceutical carrier, including a binder, a disintegrant, a lubricant, a filler, a solubilizer, a buffer, a preservative. , thickeners, compounding agents and other adjuvants.
  • “Pharmaceutically acceptable carrier”, according to various purposes of the invention, refers to any standard pharmaceutical carrier. Suitable carriers such as are known, for example, but not limited to, any of the standard pharmaceutical carriers such as phosphate buffers and various wetting agents. Other carriers may include additives for tablets, granules, capsules, and the like. Typical carriers often contain such as: starch, lotion, sugar, cellulose, some type of clay, gelatin, stearic acid or its salts, magnesium stearate or calcium stearate, talc, vegetable oils, gums, B Glycol or other known excipients. These carriers may also contain flavoring agents and coloring agents and other ingredients. The components in these carriers can be prepared by a known conventional method.
  • the mimetic of the recombinant interferon of the present invention has the AB ring and/or the BC ring structure of the recombinant interferon of the present invention (such as the specific AB ring and/or BC ring space structure described above), it is expected to be capable of The recombinant interferon of the invention is used to treat viral diseases and/or tumors.
  • the invention provides the use of a recombinant interferon crystal of the invention, an interferon mimetic or a composition comprising the above crystal or mimetic for the preparation of a medicament for the treatment of a viral disease and/or tumor .
  • the invention provides a method of treating a viral disease and/or a tumor, the method comprising administering to a subject an effective amount of a recombinant interferon crystal of the invention, an interferon mimetic or comprising the above I A composition of crystals or mimetics.
  • the invention provides a pharmaceutical composition for treating a viral disease and/or a tumor, the pharmaceutical composition comprising an effective amount of a recombinant interferon crystal of the invention, an interferon mimetic or comprising A composition of the above crystal or mimetic.
  • the viral disease can include: hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, by Epstein-Barr virus, human immunodeficiency virus (HIV), Ebo Pull virus, severe acute respiratory syndrome virus (SARS), influenza virus, cytomegalovirus, herpes simplex virus, or other types of herpes virus, papillomavirus, poxvirus, picornavirus, adenovirus, nasal Viral infection caused by virus, type I human T cell leukemia virus, or type II human T cell leukemia virus, or type III human T cell leukemia virus.
  • HIV human immunodeficiency virus
  • SARS severe acute respiratory syndrome virus
  • influenza virus cytomegalovirus
  • herpes simplex virus herpes simplex virus
  • papillomavirus papillomavirus
  • poxvirus poxvirus
  • picornavirus picornavirus
  • adenovirus nasal Viral infection caused by virus, type I human
  • the tumor is a cancer, a solid tumor
  • the tumor can include: skin cancer, Basal cell carcinoma and malignant melanoma, renal cell carcinoma, liver cancer, squamous cell carcinoma, nasopharyngeal carcinoma, solid tumor, prostate cancer, stomach/abdominal cancer, esophageal cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, shallow Bladder cancer, hemangioma, epidermoid carcinoma, cervical cancer, non-small cell lung cancer, small cell lung cancer, glioma, blood cancer, acute blood cancer, chronic blood cancer, chronic myelogenous leukemia, hairy cell leukemia, lymphoma, Multiple myeloma, erythrocytosis, Kaposi's sarcoma.
  • Example 1 Preparation of Recombinant Interferon rSIFN-co
  • This example is a method for preparing recombinant interferon rSIFN-co (stock solution) (see, in particular, the descriptions of Examples 1 and 2 of U.S. Patent No. 7,364,724 and the specification of Chinese Patent Publication No. CN1740197A The method described on pages 11-17).
  • E. coli is used to preferentially express the code.
  • the Wisconsin Package by Genetics Computer Group, Inc. Copyright 1992, Medison, Wisconsin, USA
  • molecular design of its DNA coding sequence while ensuring the amino acid sequence is unchanged, and then artificially synthesized rSIFN-co full length cDNA coding gene.
  • the two cDNA half molecules of the 5'-end 280 bp (I fragment) and the 3'-end 268 bp (II fragment) of the rSIFN-co cDNA were directly synthesized by PCR.
  • the 3'-end of fragment I and the 5'-end of fragment II have a 41 bp nucleotide sequence overlap complementary.
  • Synthesis of rSIFN-co 5'-terminal half molecule by PCR reaction I Using oligodeoxynucleotide fragment B as template, two oligodeoxynucleotide fragments of A and C as primers, PCR reaction to synthesize rSIFN of 280 bp in length -co 5'-terminal half molecule product.
  • the PCR I reaction mixture is as follows: (Unit: ⁇ 1 ) (Total volume: 50 ⁇ 1 ) Nuclease-free disinfected distilled water 39
  • a fragment primer (25 ⁇ ⁇ 1/ ⁇ 1
  • the PCR II reaction mixture is as follows: (Unit: ⁇ 1 ) (Total volume: 50 ⁇ 1 ) No nuclease disinfection of steamed water 39
  • PCR II reaction conditions and cycle were the same as PCR I.
  • the PCR-synthesized I and II fragments were assembled using the "overlap-extension PCR" method to obtain the full-length rSIFN-co cDNA full-length molecular sequence.
  • Nde I and Pst I restriction sites were introduced at the 5'-end and the 3'-end, respectively, to facilitate cloning of the rSIFN-co cDNA sequence into a plasmid vector.
  • Oligomer G 5'ATCGGCCATATGTGCGACCTGCCGCAGACCC3'
  • Oligomer H 5'ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3' (2) "Overlap-extension PCR" reaction
  • PCR reaction mixture (unit: ⁇ 1 ) (total volume: 50 ⁇ 1 ) No nuclease disinfection of distilled water 38
  • PCR product was first isolated and purified using StrataPrep PCR purification kit manufactured by Stratagen, USA, and then dissolved in sterile distilled water.
  • PCR reaction conditions and cycle were the same as the aforementioned PCR I.
  • the pLac T7 plasmid was used as a vector for the rSIFN-co cDNA gene clone.
  • the pLac T7 plasmid was transformed with pBluescript ll KS ( + ) plasmid (manufactured by Stratagen, USA).
  • the rSIFN-co full-length cDNA PCR product was purified by StrataPrep PCR purification kit (manufactured by Stratagen, USA), and then digested with Ndel and Pstl; and the pLac T7 plasmid was double-digested with Ndel and Pstl.
  • the two cleavage DNA fragments were separated by electrophoresis on 1% agarose gel, and then recovered from the gel using Promoga's Winzard DNA Purification Kit, and purified 507 bp long rSIFN-co DNA fragment and 2.9 kb plasmid enzyme. Cut the DNA fragment.
  • the two were ligated into a recombinant plasmid by T4 DNA ligase.
  • the ligation reaction mixture was transformed into DH5 ⁇ competent cells (manufactured by Gibco, USA). After overnight incubation at 37 °C, positive recombinant colonies were selected and designated as pHY-l.
  • the DNA sequencing reaction was carried out according to the instructions of the SequiThermTM Cycle Sequencing Kit (purchased from Epicentre Technologies, USA).
  • the primers were universal T7 and T3 primers, and the DNA sequencing results showed that they were consistent with the theoretical design.
  • the purified recombinant rSIFN-co protein was subjected to N-terminal 16 amino acid and C-terminal 4 amino acid sequencing. The result is:
  • Arg-Arg-Lys-Glu-COOH The rSIFN-co full-length nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 1.
  • the E. coli expression vector pBAD18 plasmid was first digested with Nde l to linearize the plasmid, and then fully digested with Xba I. After electrophoresis on 1 ⁇ 3 ⁇ 4 agarose gel, and purified by QIAEXII kit manufactured by QIAGEN, Germany, a 4.8 kb fragment of pBAD18 digested with Ndel and Xbal was obtained.
  • the pHY-1 plasmid was digested with Nde I and Xba I, and the sequence fragment of 715 bp was purified by electrophoresis on 1% agarose gel.
  • the 4.8 kb pBAD18 fragment and the 715 bp rSIFN-co and pBAD18 fragments were ligated into a recombinant plasmid under the catalysis of T4 DNA ligase.
  • the ligation reaction was transformed into DH5a competent cells, and the transformed cells were then plated on LB-Amp agar plates and cultured overnight at 37 °C.
  • a single bacterial colony was randomly picked from the above LB plate, and the recombinant plasmid strain containing the full-length coding sequence of rSIFN-co was screened by endonuclease digestion and PCR analysis.
  • One of the PCR-positive recombinant plasmids was named pHY-5.
  • the strain containing the pHY-5 plasmid was named PVIII, and after amplification, the glycerol cryopreservation solution was frozen and stored at -80 'C for use.
  • the rSIFN-co gene is regulated by the strong promoter P BAD , which in turn is regulated by the AraC protein.
  • AraC is a protein encoded by the araC gene located in the same plasmid.
  • the AraC dimer binds to 02 and 12 to form a 210 bp loop. This structure leads to complete inhibition of transcription.
  • the dimer of AraC from 02 and in turn combined with Ii and 12, the suppression of transcription.
  • Arabinose binding inactivates, inhibits, and activates transcription of the P BAD promoter, thereby stimulating P BAD to mediate high levels of rSIFN-co expression.
  • rSIFN-co can be expressed in an amount up to 50% of total bacterial proteins.
  • I grade seed bacteria were added to RM medium at a ratio of 10% (1 L contains casein 20 g, magnesium chloride 1 mmol / L (0.203 g), disodium hydrogen phosphate 4 g , 3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 37 ° C, pH 7.0, fermented to a value of about 00 600 or so, added arabinose (20%) to a final concentration of 0.02% induction, After 4 hours, the bacteria were collected and centrifuged to obtain a bacterial cell pellet;
  • renaturation treatment Dissolve the inclusion body with 6 mol/L guanidine hydrochloride (or urea) to obtain a slightly turbid denaturing solution, centrifuge at 10,000 rpm, and take the supernatant to determine the protein concentration of the denaturing solution; Mg/ml, the denaturing solution was added to the prepared reconstituted solution (0.5 mol L arginine, 150 mmol/L Tris hydrochloric acid pH 7.5, 0.2 mmol/L EDTA) in three portions, and continuously stirred at 4 ° C overnight.
  • guanidine hydrochloride or urea
  • HS cation column chromatography The column was first equilibrated with 20 mmol/L acetic acid-sodium acetate (pH 5.0), and the renaturation product obtained in step (5) was loaded at a rate of 30 ml/min. 20 column volumes of 20 mmol/L acetic acid-sodium acetate (pH 5.0), eluting the heteroprotein; followed by 5 column volumes containing 0.15 mol L sodium chloride in 20 mmol/L acetic acid-sodium acetate (pH 5.0) , eluting the heteroprotein.
  • chelating sepharoseTM fast flow HS dissociated protein solution was added to 0.2 mol/L, pH 6.6 PB buffer, and adjusted with 4 mol/L sodium chloride. 1 mol L sodium chloride, pH 6.0, ready for loading; the column was equilibrated with 1 mmol/min of 50 mmol/L disodium hydrogenphosphate buffer (pH 5.5) containing 1 mol L of sodium chloride. Speed loading; Next, elute the heteroprotein with 50 mmol/L disodium hydrogen phosphate buffer (pH 5.0); elute the heteroprotein with 50 mmol/L disodium hydrogen phosphate buffer (pH 4.0). Then, using 50 mmol/L disodium hydrogen citrate buffer (pH 3.6), the target protein is dissociated, and a chelating column is dissociated from the target protein solution;
  • HS column chromatography Dilute the chelating column to the target protein solution 30 times, and adjust the pH to 5.0, on the HS column, with 0.5 mol / L sodium chloride in PB buffer (pH 7.0 Dissociation, collection, that is, the recombinant interferon (stock solution).
  • PB buffer pH 7.0 Dissociation, collection, that is, the recombinant interferon (stock solution).
  • Recombinant interferon preparation Lyophilized injection (freeze-dried formula
  • the invention rSIFN-co stock solution 34.5 g/ml
  • Glycine 0.4mol / L Preparation process According to the formula, sterile pyrogen-free injection water solution, 0.22 ⁇ pore filter filter sterilization, stored in 6-1CTC, sampling for aseptic and pyrogen inspection after the separation of Xilin In the bottle, a single dose of 0.3-0.5 per bottle is placed in a lyophilizer and lyophilized.
  • the invention rSIFN-co stock solution 34.5 g/ml
  • Example 3 In vitro experimental study of rSIFN-co and Qianfujin anti-human breast cancer cells This example is an in vitro experimental study of recombinant interferon rSIFN-co and dried Fujin anti-human breast cancer cells.
  • the recombinant interferon rSIFN-co of the present invention and the dried hydrazine produced by Amgen Company of the present invention were used as test drugs to investigate the proliferation and apoptosis of human breast cancer MCF-7 cells and drug-resistant MCF-7/ADR cells, respectively. And the effects of oncogene expression.
  • Human breast cancer cell line MCF-7 cells and human breast cancer adriamycin-resistant strain MCF-7/ADR cells were cultured in 25 cm 2 or 75 cm 2 culture flasks respectively. After the cells were over the bottom of the flask, 0.25% trypsin was used. Digestion and passage, taking logarithmic growth phase cells for experiments.
  • rSIFNco group (0.02, 0.078, 0.313, 1.25, 5. ( ⁇ g/ml)
  • dry Fujin group 0.02, 0.078, 0.313, 1.25, 5.
  • RPMI1640 complete medium blank control group (RPMI1640 medium containing 10% calf serum (Sigma, USA), also known as RPMI1640 complete medium)
  • rSIFNco and dried hydrazine were diluted with RPMI1640 complete medium. The concentration is required (the ethanol concentration is ⁇ 1%) and stored at 4 °C.
  • MCF-7 cells and MCF27/ADR cells in logarithmic growth phase were prepared into 1.25 ⁇ 10 5 /mL cell suspension using RPMI1640 medium containing 10% calf serum, and cell viability was determined by trypan blue method > 95 %, cells were seeded on 96-well plates at 100 ⁇ per well.
  • Cell proliferation was measured by conventional MTT assay at 24 h, 48 h, and 72 h after drug addition (detection of absorbance at 490 nm on a microplate reader). Two sets of duplicate holes were set as parallel samples in each group. The above experiment was repeated 3 times, and the growth inhibition rate of the cell strain was calculated according to the following formula:
  • Proliferation inhibition rate ( ⁇ 3 ⁇ 4) (control group A value - experimental group A value) / control group A value ⁇ 100%.
  • FCM Flow cytometry
  • FCM test The cells of each group were collected for 48 hours after drug addition, and a single cell suspension was prepared.
  • the propidium iodide (PI) dye was used to determine the apoptotic rate by Elite Esp flow cytometry (Coulter, USA). The measured values are analyzed by the attached software. The above experiment was repeated 3 times.
  • rSIFN-co 5 g/mL
  • dry hydrazine 5 g/mL
  • RPMI1640 containing 10% calf serum
  • MCS-7/ADR cell culture medium was supplemented with rSIFN-co (5 g/mL), dry hydrazine (5 g/mL), and RPMI1640 containing 10% calf serum.
  • Immunohistochemistry was used to detect the expression of P53, Bcl-2, and CerbB-2 proteins.
  • the coverslips used for cell climbing were first acid-treated, washed, autoclaved, and placed in a 6-well culture plate.
  • MCF27 cells and MCF27/ADR cells grown in log phase were digested with 0.25% trypsin to form a single cell suspension, and inoculated into 6-well plates at lxlO 5 , 37 per well.
  • C ⁇ 0 2 incubate culture for 24h, after the cells were attached, each group of drugs were added.
  • the coverslips were taken out and stained by conventional immunohistochemical SABC method. The concentration of primary antibody was 1:100.
  • Outcome criterion The method of determining the staining result by reference to Volm (Volm M et al., European Journal of Cancer, 1997, 33(3): 691-693), with cell nucleus (P53), cytoplasm (Bel-2) or cell Those with yellow or brownish yellow particles in the membrane (CerbB-2) were positive. Under high magnification (400x), 5 fields of view were randomly selected for each slide, and 200 cells were counted for each field of view. The expression of each group of cells is determined by two aspects.
  • Positive staining depth According to the coloring depth of each cell, no coloring point 0 points, light yellow 1 point, brown color 2 points, brown color 3 points, the average value is the average dyeing depth of the group of cells; Percentage of positive cells: No staining was 0, stained cells ⁇ 25% for 1 point, 25 % ⁇ 50% for 2 points, > 50% for 3 points. The sum of the two, score 0 ⁇ is judged as negative, 2 ⁇ is positive, and 4-6 is strongly positive. The double-blind method was used (the stainer and the result observer did not know the grouping of the slides).
  • test data All test data were processed by SPSS 11.5 statistical software package, using t test, analysis of variance and rank correlation analysis; ⁇ 0.05, the difference was considered statistically significant.
  • MCF-7 cells rSIFN-co inhibits proliferation of MCF-7 cells.
  • the absorbance OA of each cell group with concentrations of 0.02, 0.078, 0.313, 1.25, 5.
  • MCF-7/ADR cells rSIFN-co can inhibit the proliferation of MCF-7/ADR cells.
  • the inhibition rate of rSIFN-co was higher than that of the same concentration of dry hydrazine, and the difference was statistically significant (P ⁇ 0.05). See Table 1-2.
  • the recombinant interferon rSIFN-co of the present invention and dried hydrazine produced by Amgen Corporation of the present invention were used as test drugs to observe the growth inhibition and pro-apoptotic effect on cervical cancer Caski cells (HPV16 + ) cells, respectively.
  • the drug was diluted with RPMI-1640 medium containing 10% calf serum, and cervical cancer Caski cells were cultured in a 96-well culture plate.
  • a single cell suspension was prepared by setting the number of cells in the culture medium to 1 ⁇ 10 5 /ml, and a ⁇ cell suspension was added to each well of the culture plate.
  • rSIFN-co and dry hydrazine were added to the culture plates, and the concentration gradient of each drug was 0.1564g/ml, 0.625 g/ml, 2.5 g/ml and lC ⁇ g/ml, and the control group (including 10%) was set.
  • Calf serum RPMI-1640 medium 3 replicate wells per concentration. Place in a 37 ° C, 5% C0 2 incubator for 72 hours.
  • MTT reagent (Sigma, USA) was prepared to a concentration of 5 mg/ml.
  • the wells of the culture plate were respectively added with ⁇ MTT solution, mixed gently, and placed at 37 ° C. After reacting for 4 hours in a 5% C0 2 incubator, blue crystals appeared at the bottom of the wells of the culture plate. The supernatant in the culture plate was discarded, and ⁇ dimercaptosulfoxide was added to each well, and the solution was dissolved at room temperature, and the OD value was read at a wavelength of 570 nm.
  • Cell growth inhibition rate (1 - sample well OD value / control well OD value) X 100%.
  • the first group was directly cultured for 72 hours as a control group; the 2-4 group rSIFN-co concentrations were respectively
  • the concentrations of dry ruthenium in groups 5-7 were 0.156 g/ml, 0.625 g/ml, and 2 g/ml, respectively, which were 0.156 g/ml, 0.625 g/ml, and 2.5 g/ml.
  • Each group of cells was collected and centrifuged at 1000 r/min for 5 minutes, the supernatant was discarded, and apoptosis was detected by Annexin V/PI double staining.
  • the number of cells per sample is about 1 x 10 6 , washed once with incubation buffer, centrifuged at 1000 r/min for 5 minutes, resuspended in ⁇ labeled solution, incubated for 15 minutes at room temperature in the dark, centrifuged at 1000 r/min.
  • the cells were pelleted for 5 minutes, washed once with the incubation buffer, and incubated with the fluorescent solution at 4 ° C for 20 minutes, shaken from time to time, and detected by flow cytometry.
  • rSIFN-co and dried hydrazine inhibited the growth of Caski cells, which increased with increasing concentrations of interferon.
  • the inhibitory effect of rSIFN-co on Caski cell growth at 0.625, 2.5, and 10 g/ml was higher than that of Qianfujin, and the difference was statistically significant ( ⁇ 0.01). See Table 2-1.
  • Example 5 Pharmacokinetics and Bioequivalence Study of rSIFN-co and Qianfujin This example is a study on the pharmacokinetics and bioequivalence of recombinant interferon rSIFN-co and dried hydrazine.
  • the recombinant interferon rSIFN-co of the present invention and dried hydrazine produced by Amgen Company of the present invention were respectively used as test drugs, and the pharmacokinetics and bioequivalence of the human body were compared.
  • the pharmacokinetic study of interferon in healthy humans is a difficult point in interferon research. Since the interferon level in plasma after medicinal interferon injection is a small amount, it is difficult to directly measure it in healthy adult serum by enzyme-linked immunosorbent assay (ELISA) or viral cytopathic inhibition. At present, the detection index used by the pharmacokinetics study of interferon is generally 2', 5'-OAS (2-5A oligonucleotidase), which is both an inducer of interferon and its efficacy. Representation of vitality.
  • Lyophilized recombinant high-efficiency composite interferon powder injection (experimental preparation, ie, recombinant interferon rSIFN-co, 9 ⁇ /branch).
  • Reference preparation Ganfujin injection (reference preparation, 9 g/piece), produced by Amgen Company of the United States.
  • the Eiken' Radioimmunoassay kit is supplied by Japan Eiken Chemical Co., Ltd.
  • the kit contains: (1) 1 125 - labeled 2', 5'-OAS; (2) anti-2,5,-OAS serum; (3) 2 ', 5 '-OAS standard bottles (2, 5'-OAS containing 0, 10, 30, 90, 270 or 810 pmol/dL per bottle; (4) buffer; (5) blank serum; (6) Poly(I) poly(C) agar; (7) ATP; (8) thiol ethanol; (9) accompanying quality control serum.
  • test preparations were cross-subcutaneously injected with rSIFN-co 9 g and dried Fujin 9 g in two cycles. , the cleaning period is 1 week.
  • the serum 2', 5'-OAS concentration in each blood sample was measured.
  • the paired t test was used to compare the test formulation with the reference formulation using DAS verl.O statistical software. Second, the results
  • the average enzyme concentration-time curve is plotted.
  • rSIFN-co protein single crystal is a prerequisite for determining its crystal structure.
  • the rSIFN-co sample for crystal growth was obtained from the method, technical scheme and crystalline material of the single crystal preparation of the present invention rSIFN-co rSIFN-co as described above, and its crystallographic parameters are as follows:
  • the above-mentioned rSIFN-co lyophilized powder of the present invention is dissolved in pure water and stored at a low temperature (-20 ° C).
  • the initial protein concentration was 0.42 mg/ml.
  • the rSIFN-co protein sample was concentrated to 3-3.5 mg/ml before crystallization and used immediately for crystal growth experiments.
  • the crystal cultures were carried out at room temperature (293 k ⁇ ) by hanging drop vapor phase diffusion.
  • the crystallites of rSIFN-co can appear in a variety of conditions, but it is very difficult to obtain a high quality single crystal that can be used for X-ray diffraction analysis with sufficient resolution.
  • the best crystal yield conditions for the obtained crystals were 1.2M (mol/L, the same below) Li 2 S0 4 , 0.1M CAPS ( 3-(cyclohexylamino) -1-propene Sulfonic acid), H 11.1 , 0.02 M MgCl 2 .
  • the crystal solution prepared under this condition was allowed to stand for three days to one week, and an excellent single crystal of rSIFN-co protein was obtained, and the crystal was a trigonal crystal having a crystal size of 0.42 X 0.08 X 0.08 mm.
  • the rSIFN-co protein crystal used for X-ray crystal structure analysis is shown in Figure 1.
  • Example 7 Analysis of crystal X-ray diffraction data
  • the data was collected at the synchrotron radiation station line BL5A of the Tsukuba Photonics Plant in Japan, and was completed under low temperature conditions (100K). The specific steps are as follows:
  • the wavelength of the light source is 1.0 person
  • the detector is ADSC Quantum 315 CCD (charge coupled device detector)
  • the distance from the crystal to the detector is 310mm.
  • the data is collected by the back swing method.
  • the back-swing angle used for the picture is 1°, and each exposure time is 12 seconds.
  • a total of 110 pictures are collected (see Figure 2).
  • Diffraction data processing and analysis The diffraction experiment first collects a set of intuitive diffraction images (Fig. 2). These images must be processed and analyzed using the CCP4 package for the full set of diffraction data collected for diffraction data quality assessment and structural analysis.
  • This process includes: 1) Indexing: Converting the diffraction data into crystallographic indicators (h, k, l), and calculating the unit cell parameters and space groups; 2) Parameter correction: For unit cell parameters, crystals and detectors Refinement of distance and angle and crystal inlay; 3) Integration: obtain intensity information from diffraction spots; 4) Data combination: Combine all symmetrically correlated and repeated diffraction points to produce a complete set of independent diffraction points Data; 5) Convert intensity data to structural amplitude.
  • the crystal structure of rSIFN-co was phase resolved by molecular replacement method.
  • the crystal structure (PDB number 1B5L) of sheep IFN- ⁇ (54% homology with rSIFN-co sequence) was selected as the homology structure model of molecular replacement, using software.
  • the program p haser , performs computational analysis of the rotation function and the translation function, and estimates the position and orientation of the rSIFN-co molecule in the unit cell. According to the law of Laue group and system extinction, the space group is determined as P3121, and some modifications are made to the model molecules (that is, residues 13-25, 37-69, 79-101, 114-151 in the 1B5L structure are retained).
  • the structure modification uses the program CNS 1.1.
  • the data is the parent data without phase, from which 10% is randomly extracted as the testing set, and the same set of randomly selected test sets are kept from beginning to end. All atoms in the structural model are involved in the correction, and each atom has four correction parameters, including coordinates (X, y, z) and isotropic temperature factor B.
  • the computer's automatic correction of the structure is alternated with the manual adjustment and construction of the model (using software 0).
  • the restricted NCS is used at the beginning of the correction. When the structural adjustment is basically completed, the correction is no longer used. NCS.
  • Electron density map of the model: rSIFN-co can be displayed intuitively, clearly and accurately.
  • Figure 3 shows the coordination of a representative electron density map with the structure of amino acid residues in the rSIFN-co molecule, which clearly identifies the spatial position and orientation of different amino acid residues.
  • Stereochemical index of the molecule - Lagrangian conformation The stereochemical rationality of the rSIFN-co molecule is characterized by the Lagrangian conformation (Fig. 5), and the number of amino acid residues in the optimal permitting region is 90.6%. , located at 9.1% of the permitted area, 0.4% of the general allowable area. This indicates that the rSIFN-co molecular structure model has reasonable stereochemistry.
  • rSIFN-co molecule is deposited in the unit cell.
  • An asymmetric unit of the crystal structure of rSIFN-co contains two protein molecules (called crystallographic dimers) (see Figure 7).
  • the embedding area between the dimers was 1033.3 person 2 , that is, the area contributed by each crystal was 516.6 person 2 , accounting for only about 6.4% of the total monomer area.
  • the A, B, and F faces of the A chain in the dimer correspond to the C, D, and E faces of the B chain (see figure 9)); VADAR software is used to calculate the monomer, the free energy of dimer folding is -126.9 and -257.1, respectively, that is, the free energy of dimer folding is close to the free energy of two isolated monomers (-126.9x2). This indicates a weaker interaction between the dimers.
  • there are only two weak hydrogen bonds A12 ( ARG ) NH2...NH2 B71 ( Arg ), 3.05 angstroms; A145 ( Arg ) NH1...OH B90 ( Tyr ) , 3.14 angstroms.
  • Two rSIFN-co single molecules in one asymmetric unit form a dimer.
  • Figure 8 shows the organization of the rSIFN-co crystallographic dimer.
  • the A chain consists of residues 11-103 and 111-163 (1-10, 104-110 and 164-166 are not constructed due to no representation on the electron density map); B chains are from 11-103 and 110-163
  • the residue composition (1-10, 104-109, and 164-166 was not constructed because it did not appear on the electron density map).
  • the Cys at position 29 and position 139 of each monomer form an intramolecular dike bond, and the intramolecular disulfide bond between the first and 99th Cys is due to the first Cys in the crystal.
  • residues 30-33, 47-49 of the A chain and residues 30-33 and 48-50 of the B chain are not represented by the density of the side chains, so these residues are mainly constructed as Ala or Gly.
  • residues 30-33, 47-49 position of the A chain and the 30-33' and 48-50 residues of the B chain are not expressed by the density of the side chain, so these residues are mainly constructed as Ala or Gly.
  • the structure of the two monomers is basically the same, which is related by the non-crystallographic symmetry (from B to A, polar angle Omega, Phi, Kappa are 170.64, 94.56, 118.35, respectively; the translational fractions tx, ty, tz are -1.06, respectively). -0.225, 0.155).
  • Each monomer consists of a 6-segment alpha helix (A, C, C, D, E, F), a stretch of 310 helix (B), and a linker peptide between them.
  • the folding of the entire monomer structure belongs to the helix.
  • Cytokine See Figure 9).
  • the positions of the amino acid residues corresponding to the 6-segment alpha helix (A, C, C', D, E, F) are: 13-20, 50-68, 70-76, 79-100, 114-133, 138- 160.
  • the position of the amino acid residue corresponding to a stretch of 310 (B) is 40-43.
  • Figure 9 clearly shows the distribution and organization of these secondary structures.
  • Figure 10 shows the correspondence between the secondary structure and the amino acid sequence.
  • Example 11 Three-dimensional structure of rSIFN-co and IFN-a2b
  • Interferon can be divided into two categories according to the receptor: Type I and Type II.
  • Type I groups can be further divided into a, ⁇ , ⁇ , etc., wherein IFNa includes nearly 15 different subtypes, and the protein sequence homology between different a class proteins is above 80%, but their functions show Diversity.
  • the rSIFN-co protein belongs to the non-natural artificially designed protein in IFNa. So far, type I IFNs have only six three-dimensional structures (Table 5), and their amino acid sequence homology shows sequence alignment (as shown in Figure 11).
  • RMSD RMSD
  • rSIFN-co contains two molecules in the asymmetric unit, and the crystallographic asymmetric unit of IFN-a2b has six protein molecules composed of three dimers.
  • the dimeric organization of rSIFN-co is significantly different from that of IFN-a2b (as shown in Figure 13).
  • interferon as a cytokine first binds to a specific receptor on a cell membrane to activate various signaling pathways, and the body produces biological effects such as antiviral and antitumor.
  • rSIFN-co belongs to IFN- ⁇ -like interferon, and its receptor on the cell membrane is known to be composed of IFNAR1 and IFNAR2, and thus a three-dimensional structural model of binding of the receptor to IFN-a molecule is constructed (as shown in Fig. 15a). ).
  • rSIFN-co interferon has a similar molecular skeleton to IFN-a2b, it has a significantly different structure in its functionally active region. Therefore, from the molecular structure closely related to pharmacological activity, rSIFN-co is a new type of molecule different from IFN-a2b, resulting in significantly different biological and pharmacological characteristics between the two. Interferon-rSIFN-co may have unique physiological and pharmacological effects based on the three-dimensional structure of the characteristic key parts. Table 6
  • V 6C - 1 00 • ⁇ S99 ⁇ - ⁇ 9 ⁇ • ⁇ ⁇ V OHV ZD 9Z WO V
  • V 6f •8S 00 ⁇ 09 ⁇ ⁇ 6 ⁇ - 60 ⁇ •81 ⁇ 8 ⁇ - ⁇ V DHV V3 IZ WOIV
  • V ⁇ 9 mesh M V ⁇ 33 O/0ZAVu/u.0U/losd ssoio
  • V 69 •6 00 " ⁇ m ⁇ 8 ⁇ - ⁇ 6 ⁇ IS ZZ ⁇ •9 9 V V1V 3 fLf WOXV
  • V ZO OS 00 " ⁇ 6 6 ⁇ - 6 ⁇ '9 ⁇ ⁇ 9 ⁇ ' ⁇ - 9 ⁇ V N 66Z Dish V
  • V 'Of 00 " ⁇ 99 ⁇ ⁇ 8 ⁇ - OZI '82 80 ⁇ - fZl V HHd zao WL WOIV
  • ATOM 862 C GLY A 133 -35. 929 42. 684 - 12, 592 1. 00 62. 83 A
  • ATOM 876 C ALA A 135 -41. 221 40. 096 -12. 501 1. 00 61. 65 A
  • ATOM 882 CD1 TYR A 136 -40. 241 37. 378 -10. 807 1. 00 62. 66 A
  • ATOM 1251 CA GLY B 33 -64. 646 20. 645 -51. 009 1. 00 89. 82 B
  • ATOM 1270 C ASP B 35 -64. 579 26. 767 -45. 784 1. 00 73. 69 B

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Abstract

本发明提供了一种重组干扰素（rSIFN-co）晶体，所述重组干扰素具有（i）与人共有干扰素相同的氨基酸序列，以及（ii）与IFN-α2b相比改变了的三维结构。本发明的重组干扰素具有增强的生物活性。本发明还提供了一种可用于药物筛选和/或药物设计的所述重组干扰素的结构模型及该重组干扰素的模拟物。

Description

空间构象改变的重组干扰素的晶体、 其三维结构及应用 技术领域
本发明主要涉及空间构象改变的重组干扰素的晶体、其结晶方法、其三维结 构、 所述晶体和三维结构的应用以及所述重组千扰素的模拟物。 背景技术
干扰素 （IFN )是一种由多种细胞产生的可溶性蛋白质， 其具有多种重要的 生物功能， 包括抗病毒、 抗肿瘤和免疫调节功能。 根据产生细胞类型、 受体、 生 物活性等的不同， 干扰素可分为 I型、 II型和 III型干扰素。 I型干扰素主要由病 毒及合成的双链 RNA所诱导， 因此也称为抗病毒干扰素。 现有三种类型的 I型 干扰素： IFNa、 INFp和 IFNoo。 II型干扰素也称为免疫干扰素或 IFNy, 由 T细 胞产生， 并且在体内是一种重要的免疫调节因子。 III型干扰素则由 IFN-λ分子 构成。
如大量的相关专利和公开文献所显示的， 近年来，世界上许多公司都在参与 干扰素的研究。举例来说， 美国专利第 4,695,623号和第 4,897,471号公开了新型 人干扰素多肽，其氨基酸序列包含存在于天然 a-干扰素多肽中的共同或占优势的 氨基酸。 该新型干扰素多肽被命名为 IFN-con (共有干扰素 a )。 所公开的氨基酸 序列被命名为 IFN-conl、 IFN-con2和 IFN-con3。 还公开了编码 IFN-cons的基因 以及在大肠杆菌中的基因表达。研究表明与白细胞干扰素或其它 I型干扰素相比， 该重组 IFN-con具有更高的体外抗病毒、 抗增殖及天然杀伤细胞活性。
美国专利第 5,372,808号公开了人 IFN-con在疾病治疗中的用途。 与之前临 床审批通过的 a-干扰素如先灵葆雅公司生产的干扰能@ ( IFN-a2b, SGP )相比， 重组人共有干扰素显示具有更低的副作用。 1997 年底， 美国食品与药品管理局 ( FDA )批准了美国安进（Amgen )公司生产的人共有干扰素作为丙型肝炎临床 治疗用药， 商品名为干复津（ INFERGEN® , interferon alfacon- 1 )。
美国专利第 7,364,724号和中国专利公开号 CN1740197A (其全文在此并入 作为参考）均公开了同一种功效增强、副作用更小且能够大剂量使用的重组干扰 素（在下文中也称为 "rSIFN-co" )。.所述重组干扰素具有与干复津相同的氨基 酸序列， 但其空间构象和生物效力发生了变化。 此外， 虽然上述中国专利公开号 CN1740197A中还公开了所述重组干扰素的晶体及其制备方法，但所得到的晶体 质量不好， 晶体内部结构疏松， X-射线衍射分辨率低， 仅达到 5人， 无法适用于 进一步的蛋白质空间结构解析，以获得有用的结构信息。因此，获得质量良好的、 X-射线衍射分辨率高的所述具有改变的结构和功能的重组干扰素的晶体，测定其 三维结构、建立模型并通过利用所述结构和模型来开展药物设计以及改良已有干 扰素的效力是非常有意义的。 发明概述
本发明涉及上述美国专利第 7,364,724 号和中国专利公开号 CN1740197A所 公开的重组干扰素的晶体， 这样的重组干扰素包含 SEQ ID ΝΟ:1所示的氨基酸 序列。 进一步地， 本发明提供了上述重组干扰素的结晶方法、 包含所述晶体的组 合物。 此外， 本发明提供了所述重组干扰素不同于现有技术已公布的 IFN- oc 2b 等干扰素的三维结构、 也不同于美国安进公司干复津的计算模型结构的三维结 构、所述三维结构在基于计算机的鉴定能与干扰素相互作用的候选化合物、设计 干扰素模拟物、 进行合理药物设计中的应用。 更进一步地， 本发明提供了所述重 组干扰素的模拟物、 包含所述模拟物的组合物以及所述晶体、模拟物或组合物在 制备用于治疗病毒性疾病和 /或肿瘤的药物中的应用。 附图简述
图 1显示了用于晶体结构分析的本发明的重组干扰素 （rSIFN-co ) 的 单晶。
图 2显示了 rSIFN-co晶体的 X-射线衍射图 （分辨率为 2.6 A )。
图 3显示了 rSIFN-co分子晶体结构中局部 2Fo-Fc的 Ι .Οσ电子密度图。 图 4显示了 rSIFN-co的所有原子的平均温度因子随残基的分布图；其 中图 4a为 A链的图； 图 4b为 B链的图。
图 5显示了 rSIFN- co蛋白质分子结构模型中所有氨基酸残基的 （ φ , ψ ) 值在拉氏构象图上的分布， 此图参考了分辨率至少 2.0 埃、 R 因子低于 20%的 118个结构，在最佳区域内有 90%以上是高质量的模型，其统计数据如下： 统计数据
在最适区域内的残基 [ A、 B、 L ] 240 90.6% 在附加允许区域内的残基 [ a、 b、 1、 p ] 24 9.1% 一般允许区域的残基 [ ~ a、 ~ b、 ~ 1、 ~ p ] 1 0.4%
在不允许区域内的残基 0 0.0% 除甘氨酸和脯氨酸以外的残基数 265 100.0% 末端残基数（甘氨酸和脯氨酸除外） 127
甘氨酸残基数 18
脯氨酸残基数 6 总残基数 416 图 6显示了 rSIFN-co晶胞堆积图。
图 7显示了 rSIFN-co二聚体结构的组装结构对。
图 8显示了 rSIFN-co晶体学二聚体的组织 （图 8a, 图 8b ) 及（ 碳原 子的均方根偏差 （RMSD , 方框代表缺失残基）（图 8c )。
图 9显示了 rSIFN-co单分子的结构（仅示出主肽链）； 其中图 9a为侧 视图； 图 9b为俯视图； 图 9c为拓朴示意图； 图 9d为二级结构的拓朴组 织方式。
图 10显示了 rSIFN-co二级结构与氨基酸序列匹配图； 其中灰线方框 代表在结构中未搭建的氨基酸残基， 蓝线方框代表在结构中构建为 Ala或 Gly的氨基酸残基， 连线为两对二硫键， 下标绿色为结构中已构建的 1对 二硫键。
图 11显示了 rSIFN-co蛋白与同源 IFN多肽的序列对比图。
图 12显示了 rSIFN- co与 IFN-oc2b的 结构比较差异图。
图 13显示了 rSIFN-co (红色） 与 IFN-oc2b (黄色） 二聚体叠合图。 图 14显示了 rSIFN-co三维结构与美国安进公司 Infergen的计算模型结 构的比较差异图。
图 15显示了 （ a ) IFN-oc类蛋白与受体的结合模型； （b ) IFN-o 类功 能区示意图 （蓝圈区为重要功能区）。
图 16显示了 18名受试者皮下注射 rSIFN-co 9 μ g和 INFERGEN® 9 μ g 后的均值血中酶浓度 -时间曲线。 发明详述
以下所提供的是帮助本领域技术人员实施本发明的本发明详细描述。
重组千扰素 (rSIFN-co )
本发明的重组干扰素在结晶之前的纯化产物是根据美国专利第 7,364,724 号说明书实施例 1和 2和 /或中国专利公开号 CN1740197A说明书中第 11- 17页记载的方法制得的。 关于本发明的重组干扰素的限定， 可具体参见 美国专利第 7，364，724 号和 /或中国专利公开号 CN1740197A。 在一个实施方案 中， 本发明的重组干扰素的氨基酸序列以及编码所述氨基酸的核苷酸序列 分别如下所示：
M C D L P Q T H S L G N R R A L I IJ A
1 ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCT
TACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA
Q M R R I S P F S C L K D R H D F G F P
61 CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCG
GTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC
Q E E F D G N Q F Q K A Q A I S V L H E
121 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAA
GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCA AGACGTGCTT
M I Q Q T F N L F S T K D S S A A W D E
181 ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAA
TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT
S L L E F Y E L Y Q Q L N D L E A C
241 TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGC
AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG AC TGCTGGA CCTTCGAACG V I G E V G V E E T P L M N V D S I L A 301 GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCT
CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA
V K K Y F Q R I T L Y L T E K K Y S P C
361 GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGC
CAATTTTTTA TGAAGGTCGC ATAGTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG
A W E V V R A E I M R S F S L S T N L Q
421 GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG
CGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC
E R L R R K E ( SEQ ID NO : l )
481 GAACGTCTGC GTCGTAAAGA ATAA ( SEQ ID NO : )
CTTGCAGACG CAGCATTTCT TATT ( SEQ ID NO : 3 ) 此外， 本发明的重组干扰素在 190-250nm范围内的圆二色谱显著不同 于在同样条件下测得的干复津的圆二色谱， 在 250-320nm范围内的圆二色 谱与干复津的圆二色谱也显著不同 （参见中国专利公开号 CN1740197A说明 书第 3页第 22-25行、 实施例三和图 6A-D )。
并且，本发明的重组干扰素的三维结构既不同于现有技术已公布的 IFN- cc 2b的三维结构（参见图 12 ),也不同于干复津的计算模型结构（参见 KORN, AP等， Journal of Interferon Research 1994, 14: 1-9 ), 由两者比较结果尤其 可见二者的 AB环有着明显的区别，其 BC环也无法完全重合（参见图 14 )。
另外， 本发明的重组干扰素在肌肉注射给体重指数 （BMI ) 为 18-23 范围内的人体后，以采血时间对受试人体血清中的 2-5 A寡聚核苷酸酶（也 称为 "2'，5'-OAS" ) 浓度作图， 所得曲线一般表现为双峰， 其曲线下面积 显著大于在同一条件下注射干复津所获得的曲线下面积。 此外， 本发明的 重组干扰素在注射给人体后其在人体内的半衰期比干复津在人体内的半 衰期更长。
已进行的实验的结果还证实， 本发明的重组干扰素具有比目前投入临 床使用的其它任何干扰素 （包括干复津） 更强的效力。 例如， 对于乙型肝 炎病毒而言， 本发明的重组干扰素不仅能抑制乙型肝炎病毒的 DNA 复制 而且还能抑制乙肝病毒表面抗原 （HBsAg ) 和 e抗原 （HBeAg ) 的分泌， 抑制乙肝病毒核心抗原（ HBcAg ) DNA复制的效力与千复津相比提高约一 倍。 本发明的重组干扰素的体外药效学显示它不仅能抑制乙型肝炎病毒的 DNA复制， 而且能抑制表面抗原和 e抗原的分泌， 其细胞学毒性仅为临床 现用干扰素的 1/8， 但抗病毒活性却是临床现用干扰素的 5-20倍， 同时在 人体内具有更高更广 i普更长时间的生物学应答反应。
此外， 无论是就病毒性疾病的预防还是就肿瘤的治疗而言， 所述干扰素都具 有与其它干扰素 （包括干复津）相比， 更高的抗病毒活性和更低的副作用。 例 如， 本发明的重组干扰素不但具有强于临床现用干扰素 20 倍的抗病毒活 性， 以及明显强于重组人 α 型干扰素 （包括干复津） 的抗肿瘤如抗乳腺癌 和宫颈癌作用； 还极大地降低了毒副作用并可安全的大剂量使用 （每剂用 量可〉 1000 万 IU ) ， 使需要大剂量用药干扰素的部分病毒性疾病或肿瘤 的成功治疗成为可能。
由此可见， 本发明所使用的重组干扰素具有与干复津不同的空间构 象、 增强的生物学活性以及不同的药代动力学特性。
当在本文中使用时， 术语 "空间构象" 、 "空间结构" 、 "三维结构" 和 "三维构象" 可互相交换使用。
因此， 在一个实施方案中， 本发明所使用的重组干扰素包含 SEQ ID ΝΟ: 1 所示的氨基酸序列，并由包含 SEQ ID NO: 2的核苷酸序列编码。进一步地， 本发明所使用的重组千扰素的氨基酸序列如 SEQ ID ΝΟ: 1 所示， 并由如 SEQ ID NO: 2所示的核苷酸序列编码。 进一步地， 所述重组干扰素与具有 如 SEQ ID ΝΟ: 1所示的氨基酸序列 （或与具有相同的氨基酸序列）但不为 如 SEQ ID NO: 2所示的核苷酸序列编码的干扰素例如干复津相比， 具有改 变的空间构象和 /或增强的生物学活性和 /或不同的药代动力学特性， 举例来说， 所述重组干扰素具有改变的空间构象和增强的生物学活性、具有改变的空间构象 和不同的药代动力学特性、 或具有增强的生物学活性和不同的药代动力学特性。 更进一步地， 所述改变的空间构象包括： 所述重组干扰素在 190-250nm 范围内 的原二色谱显著不同于在同样条件下测定的千复津的圆二色谱和 /或在 250-320nm范围内的圆二色谱与干复津的圆二色谱也显著不同；所述增强的 生物学活性包括： 增强的抗病毒活性、 增强的抗肿瘤活性、 更低的副作用和 /或 能大剂量使用， 例如每剂用量可〉 1000万 IU, 举例来说， 所述增强的生物 学活性可以是增强的抗病毒活性和增强的抗肿瘤活性等等， 进一步地所述肿瘤 可以是乳腺癌和宫颈癌； 所述不同的药代动力学特性包括： 在肌肉注射给体重 指数为 18-23范围内的人体后，以采血时间对受试人体血清中的 2-5 A寡聚 核苷酸酶浓度作图， 所得曲线的曲线下面积显著大于在同一条件下注射干 复津所获得的曲线下面积和 /或与千复津相比更长的半衰期。
在另一个实施方案中， 本发明中使用的重组干扰素可由如下方法所产生， 所述方法包括：将编码重组干扰素的包含 SEQ ID NO: 2的核苷酸序列导入 分离的宿主细胞中， 在适合于表达所述重组干扰素的条件下培养所述宿主 细胞， 和收获所述重组干扰素， 其中所述重组干扰素具有 SEQ ID ΝΟ: 1的 氨基酸序列， 并且所述重组干扰素抑制乙型肝炎病毒的乙肝病毒表面抗原 ( HBsAg )和 e抗原 （HBeAg ) 的分泌。 进一步地， 所述宿主是大肠杆菌， 如。 大肠杆菌 LGM 194。 进一步地， 其中包含 SEQ ID NO: 2的核苷酸序 列处于启动子 P^D的控制下。 进一步地， 其中收获步骤包括从培养发酵液 中提取干扰素， 收集包涵体， 所收获干扰素的变性和复性。 更进一步地， 所述收获步骤进一步包括分离纯化所述重组干扰素 （参见美国专利第 7,364,724 号权利要求） 。 重组千扰素晶体及结晶方法
重组干扰素晶体
本发明提供了一种本发明重组干扰素的晶体。
在一个实施方案中， 本发明提供了一种包含 SEQ ID ΝΟ:1所示的氨基酸序 列的重组干扰素的晶体。 进一步地， 所述晶体具有三方晶型。 在一个实施方案 中， 所述晶体的空间群为 Ρ3 1。在某些实施方案中， 所述晶体的单晶胞参数为： a=b=77.92埃， c=125.935埃， α = β =90°， γ =120°, 所有晶胞参数的可变性不超 过 5%。 在某些实施方案中， 所述晶体在一个不对称单位中包含 2个分子。 在某 些实施方案中， 所述晶体包含共价或非共价结合的金属离子。 进一步地， 所述金 属离子可以是镁离子、锌离子等， 这些金属离子可介导晶体中干扰素二聚体的形 成。 在某些实施方案中， 所述重组干扰素由包含 SEQ ID NO: 2的核苷酸序列所 编码。
在更进一步的实施方案中， 本发明提供了一种包含 SEQ ID ΝΟ: 1所示的氨 基酸序列 （优选其氨基酸序列如 SEQ ID ΝΟ: 1所示） 的重组干扰素的晶体， 其 中该晶体的空间群为 P3 1 , 在一个不对称单位中包含 2个分子， 以及单晶胞参 数为： a=b=77.92埃， c=125.935埃, α=β=90°, γ=120°, 所有晶胞参数的可变性 不超过 5%。 进一步地， 这样的重组干扰素由包含 SEQ ID NO: 2的核苷酸序列 所编码， 优选由如 SEQ ID NO: 2所示的核苷酸序列所编码。
结晶方法
本发明提供了制备（培养）本发明重组干扰素晶体的方法。
在一个实施方案中， 本发明提供了一种制备（培养）本发明重组干扰素晶体 的方法， 包括步骤： 将重组干扰素浓缩至约 3-3.5mg/ml , 置于包含 Li2S04、 CAPS
( 3- (环己氨基 ) -1-丙磺酸 )和 MgCl2的结晶溶液条件下合适的一段时间， 获得 所述晶体。 进一步地， 所述晶体培养方法是在室温如 293K下进行的。 在某些实 施方案中， 所述晶体培养采用悬滴法或坐滴法进行的， 优选采用悬滴法（也称为 悬滴汽相扩散法）进行。在某些实施方案中，所述结晶溶液条件包含约 1.0-约 1.5M Li2S04、 约 0.05-约 0.15M CAPS ( 3- (环己氨基 ) -1-丙磺酸 )和约 0.01-约 0.03 M MgCl2。在某些实施方案中，所述结晶溶液条件的 pH值在约 10.5-约 12.0范围内， 优选约 11.1。在某些实施方案中，所述结晶溶液条件为 1.2M Li2S04、 0.1M CAPS
( 3- (环己氨基） -1-丙磺酸）， pH l l . l , 0.02 M MgCl2。 在某些实施方案中， 所 述晶体培养包括将上述含重组干扰素的结晶溶液静置约 1天-约 2周， 优选约 2 天-约 10天， 更优选约 3天到约 1周， 例如 3天 -1周。 X-射线结晶学分析 在此公开的干扰素的每一个组成氨基酸都通过一组结构坐标（也称为 "原子 坐标" ）得到定义。 术语"结构坐标"指由数学方程式导出的笛卡儿坐标， 该数学 方程式与 X-射线单色光通过以晶体形式存在的本发明的干扰素的原子（散射中 心）衍射获得的图样相关。衍射数据用于计算晶体重复单元的电子密度图。然后， 电子密度图用于建立干扰素蛋白或蛋白 /配体复合物的单个原子的位置。
通过数学方式操作所述干扰素或者干扰素 /配体结构坐标， 还可产生轻微的 结构坐标改变。 举例来说， 在此公开的结构坐标可通过如下手段进行操作： 结构 坐标的结晶学置换、 结构坐标的分段、 结构坐标集合的整体加减、 结构坐标的翻 转， 或上述手段的任意组合。 而由于氨基酸突变、 添加、 取代和 /或缺失或在任 何组成部分中的其他改变而导致的晶体结构的改变， 也可引起结构坐标的变化。 在个体坐标中这样的轻微变化对总体形状影响不大。如果与原始坐标相比这样的 改变处于可接受的标准误差之内， 则所得到的三维形状被认为是结构等价的。
应当指出本发明干扰素的个体结构坐标中的轻微变化预计不会明显改变上 述实体如可与干扰素或其部分（如其 AB或 BC环）结合的配体的特性。 当在本 发明中使用时， 本发明重组干扰素的 "AB环" 指本发明重组干扰素 （其氨基酸 序列如 SEQ ID NO: 1所示）第 25-33位氨基酸残基，即该 AB环的序列为 SEQ ID NO:4所示的氨基酸序列 SPFSCLKDR; 本发明重组干扰素的 "BC环" 指本发 明重组干扰素（其氨基酸序列如 SEQ ID ΝΟ:1所示）第 44-52位氨基酸残基， 即 该 BC环的序列为 SEQ ID NO:5所示的氨基酸序列 DGNQFQKAQ。在上下文中， 短语"与 ......结合"指配体或其部分与干扰素分子或其部分之间相邻近的情况。这 样的结合可以是非共价的， 其中这样的相邻近在能量上受益于氢键、 范德华力或 静电相互作用； 所述结合也可以是共价的。 因此， 作为实例， 与干扰素的结合口 袋或结合区结合的配体也应预期能结合结构等价的结合口袋或结合区，或与之相 互作用。
对于本发明而言，对于任何分子或分子复合物或其任意部分， 当叠合到在此 描述的相关骨架原子上，具有小于约 0.65埃的保守残基骨架原子（如 N、 Ca、 C、 0， 优选 Ca )均方根偏差时， 被认为是"结构等价的"。 换言之， 这两个分子的这 些部位的晶体结构在可接受的误差范围内是基本上相同的。特别优选的结构等价 的分子或者分子复合物为具有如下限定的分子或者分子复合物：在此公开的结构 坐标的完整集合 ±'j、于约 0.65埃的来自于那些氨基酸的保守骨架原子的均方根偏 差。 更优选地， 所述均方根偏差最多约 0.5埃， 还更优选最多约 0.35埃。 本发明 的其他实施方案包括具有如下限定的分子复合物： 在此公开的 AB或 BC环的结 构坐标 ±小于约 0.65埃的均方根偏差， 所述均方根偏差优选最多约 0.5埃， 还更 优选最多约 0.35埃。
术语"均方根偏差"指偏差的平方的算术平均值的方根，其用于表示与趋势或 对象的偏差或变化。 在一个实施方案中， 所述"均方根偏差"规定了一种蛋白质骨 架相对于如在此描述的结构坐标所限定的干扰素骨架或其部分的变化。
X-射线结构坐标定义了空间中点的独特构象。 本领域技术人员应当理解蛋 白、 或蛋白 /配体复合物、 或它们的一部分的结构坐标定义了一系列相应的点； 而这些点又继而定义了三维构象。 如果坐标之间的距离和角度保持基本上相同， 则相似或相同的构象可为一组完全不同的坐标所定义。此外， 只要保持角度基本 上相同， 可伸缩的点构象就可通过在坐标之间增加或减少距离来定义。
各种计算分析可用于确定分子或其部分与在此公开的干扰素或其部分是否 是根据三维结构定义的"结构等价的"。 举例来说， 可通过各种计算分析进行不同 结构、相同结构的不同构象、 或相同结构的不同部分之间的比较。 在一个实施方 案中， 所述分析可包括以下四个步骤： （1 )加载需要比较的结构； （2 )在这些结 构中定义原子等价性； （3 )执行拟合操作； 和（4 )分析结果。 重组千扰素 (rSIFN-co )的三维结构
本发明提供了本发明重组干扰素的三維结构。
这种三维结构既不同于现有技术已公布的 IFN- a2b的三维结构（参见图 12 )、 也不同于美国安进公司干复津的计算模型结构 （参见图 14 ) , 尤其在 AB环和 BC环上与 IFN- a 2b及干复津存在着结构上的差异。
在一个实施方案中，所述重组干扰素的三维结构包含如表 7所示的重组干扰 素的原子坐标， 所述原子坐标任选地在小于约 0.65埃，优选约 0.5埃以及更优选 约 0.35埃的保守残基骨架原子优选 Ca原子（也称为 "a碳原子" ） 的均方根偏 差内变化。
在一个实施方案中，在上述重组干扰素的三维结构中， 所述重组干扰素的每 个单体都由 6段 α螺旋、 1段 310螺旋及其间的连接肽段构成。 所述 6段 α螺旋 对应的氨基酸残基位置分别为 13-20、 50-68、 70-76、 79-100、 114-133和 138-160； 所述的 1段 31()螺旋对应的氨基酸残基位置为 40-43。 单体结构的折叠方式属于 螺旋状细胞因子型（helical cytokine ), 特征在于： 在将所述重组干扰素的 α碳原 子骨架与 IFN-a2b蛋白的 a碳原子骨架以最小二乘法方式进行叠合后，该重组干 扰素在第 25-33位残基（ AB环）上的各 a碳原子与所述 IFN-a2b蛋白在对应位 残基上的各 碳原子的位置均方才艮偏差 ( location root-mean-square deviation ) 为 3.63埃 ±5%。
优选的是， 所述重组干扰素与所述 IFN-a2b蛋白在第 25位残基上的 α碳原 子的位置均方根偏差为 3.291埃 ±5%; 在第 26位残基上的 α碳原子的位置均方 根偏差为 4.779埃 ±5%; 在第 27位残基上的 α碳原子的位置均方根偏差为 5.090 埃 ±5%; 在第 28位残基上的 α碳原子的位置均方根偏差为 3.588埃 ±5%; 在第 29位残基上的 a碳原子的位置均方根偏差为 2.567埃 ±5%; 在第 30位残基上的 α碳原子的位置均方根偏差为 2.437埃 ±5%;在第 31位残基上的 α碳原子的位置 均方根偏差为 3.526埃 ±5%; 在第 32位残基上的 α碳原子的位置均方根偏差为 4.820埃 ±5%; 在第 33位残基上的 α碳原子的位置均方根偏差为 2.756埃 ±5%。
还优选的是， 所述重组千扰素在第 44-52位残基（ BC环）上的各 (X碳原子 与所述 IFN-a2b蛋白在对应位残基上的各 α碳原子的位置均方根偏差为 2.90埃 ±5%。 其中， 所述重组干扰素与所述 IFN- oc2b蛋白在第 44位残基上的 碳原子 的位置均方根偏差为 1.614埃 ±5%， 在第 45位残基上的 oc碳原子的位置均方根 偏差为 1.383埃 ±5% , 在第 46位残基上的 碳原子的位置均方根偏差为 2.735 埃 ±5% , 在第 47位残基上的 oc碳原子的位置均方根偏差为 2.709埃 ±5% , 在第 48位残基上的 α碳原子的位置均方根偏差为 5.018埃 ±5% , 在第 49位残基上的 α碳原子的位置均方根偏差为 4.140埃 ±5% , 在第 50位残基上的 a碳原子的位 置均方根偏差为 3.809埃 ±5% , 在第 51位残基上的 α碳原子的位置均方根偏差 为 2.970埃 ±5% ,在第 52位残基上的 α碳原子的位置均方根偏差为 0.881埃 ±5% (上述所列的"位置均方根偏差"均为坐标位置的均方根偏差）。
在另一方面， 本发明提供了经选择的本发明重组干扰素的三维结构的一部 分， 其中所述三维结构经选择的部分包含表 7中所包含的来自第 25-33位氨基酸 残基和 /或第 44-52位氨基酸残基的一个或更多个氨基酸残基的原子坐标。 在某 些实施方案中， 本文所述的 "一个或更多个氨基酸残基" 包括 1、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18 个氨基酸残基。 在某些实 施方案中， "所述三维结构经选择的部分" 包含表 7中所包含的来自第 25- 33位 氨基酸残基和 /或第 44-52位氨基酸残基的原子坐标。 在某些实施方案中， "所述 三维结构经选择的部分" 包含表 7中所包含的来自至少 5、 6、 7、 8、 9、 10、 11、
12、 13、 14、 15、 16、 17、 18、 19、 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、
30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41、 42、 43、 44、 45、 46、 47、
48、 49、 50、 51、 52、 53、 54、 55、 56、 57、 58、 59、 60、 61、 62、 63、 64、 65、
66、 67、 68、 69、 70、 71、 72、 73、 74、 75、 76、 77、 78、 79、 80、 81、 82、 83、
84、 85、 86、 87、 88、 89、 90、 91、 92、 93、 94、 95、 96、 97、 98、 99、 100个 氨基酸残基的原子坐标。在某些实施方案中， 上述原子坐标任选地在小于约 0.65 埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Ca原子的均方 根偏差内变化。
在又一方面，本发明提供了一种包含本发明的重组干扰素的三维结构的蛋白 质空间结构模型。在一个实施方案中， 所述蛋白质空间结构模型可以是电子密度 图、 线框模型、 网状模型、 空间填充模型、 棍状模型、 带状模型、 分子表面模型 等等。
在再一方面， 本发明提供了一种可伸缩（scalable )的由点组成的三维构象， 所述点的至少一部分来源于在此披露的结构坐标，或来源于包含本发明的重组干 扰素的 AB环或 BC环的肽。 在一个实施方案中， 所述可伸缩的由点组成的三维 构象展示为全息图、 立体图、 模型或者计算机显示图片。 三维结构的应用
筛选 /设计与重组干扰素相互作用的候选物质
在一方面， 本发明提供了一种筛选 /设计与本发明重组干扰素相互作用的候 选化合物的方法。 进一步地， 所述方法利用到本发明重组干扰素的三维结构， 更 进一步地， 所述方法是基于计算机的。 在一个实施方案中， 本发明提供了一种基 于计算机的鉴定能与重组干扰素相互作用的候选化合物的方法，所述方法包括步 骤： （a )提供包含表 7所示的重组干扰素的原子坐标的三维结构， 所述原子坐标 任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原 子优选 Ca原子的均方根偏差内变化； 和（b )选择具有能与上述三维结构或该 三维结构经选择的部分相互作用的结构特征的候选化合物，从而答定能与所述重 组干扰素相互作用的候选化合物。在某些实施方案中， 所述结构特征选自抗原性 位点、 亲水特性、 表面可接近性和结构基序。 在某些实施方案中， 步骤（b ) 中 的候选化合物的选择和鉴定包括： （ i )对多个候选化合物生成三维结构； 和（ ii ) 将步骤（i ) 的三维结构各自与上述步骤 ) 中的三维结构或该三维结构经选择 的部分进行拟合， 以发现在能量上最有利的相互作用，从而鉴定能与所述重组干 扰素相互作用的候选化合物。在某些实施方案中，所述方法进一步包括步骤：（c ) 获取或合成候选化合物； 和（d )使所述候选化合物与所述重组干扰素接触以确 定该候选化合物与所述重组干扰素相互作用的能力。进一步地， 所述确定候选化 合物与重组干扰素相互作用的能力的步骤还可包括进一步包括测定所述重组干 扰素与候选化合物接触时的活性。 举例来说， 要被测试的干扰素活性包括： 抗病 毒活性、 抗肿瘤活性、 抗增殖活性、 天然杀伤细胞活性和免疫调节活性。 在某些 实施方案中， 所述候选化合物是与所述重组干扰素或其经选择的部分结合的配 体。 举例来说， 所述配体选自： 受体、 改性剂、 激动剂和拮抗剂， 举例来说， 这 样的受体可以是 IFNAR1、 IFNAR2或它们的复合物； 以及其中所述经选择的部 分包含所述重组干扰素的第 25-33位氨基酸残基（ AB环的序列 )和 /或第 44-52 位氨基酸残基（BC环的序列） 的一个或更多个氨基酸残基。 进一步地， 其中所 述经选择的部分包含来自所述重组干扰素的第 25-33位氨基酸残基和 /或第 44-52 位氨基酸残基。
在另一方面，本发明提供了一种鉴定能与本发明重组干扰素相结合的潜在配 体的方法。 在一个实施方案中， 所述方法包括以下步骤： 将在此公开的晶体暴露 于一种或多种包含潜在配体的样品， 并确定是否形成配体-干扰素分子复合物。
在另一方面，本发明提供了一种获取用于设计能与干扰素形成分子复合物的 潜在配体的结构信息的方法。 在一个实施方案中， 所述方法包括以下步骤： 将在 此公开的晶体暴露于潜在配体库， 并确定是否形成配体 -干扰素分子复合物。
在另一方面， 本发明提供了一种计算机辅助的用于鉴定、设计或制备潜在干 扰素活性调节剂的方法。在一个实施方案中， 所述方法包括筛选化学或者生物实 体库。
本领域技术人员可应用结晶学手段来筛选并鉴定可能成为干扰素配体的化 学或生物实体（见美国专利第 6，297，021号）。 举例来说，一种优选的方法可包括 以下步骤： 获得非配体化的干扰素晶体； 将所述非配体化的干扰素晶体暴露于一 种或多种包含潜在干扰素配体的测试样品； 和确定是否形成配体 -干扰素分子复 合物。 可通过多种方法将所述干扰素暴露于潜在配体， 所述方法包括但不限于， 将干扰素晶体浸泡于含有一种或多种潜在配体的溶液中， 或者，在一种或多种潜 在配体的存在下共结晶干扰素。
来自所述配体-干扰素复合物的结构信息可优选地用于设计新配体， 该新配 体较已知配体结合得更紧密、 结合得更特异， 具有期望的生物活性特性， 具有更 高的安全性， 甚至具有以上优点的结合。 举例来说， 计算出的电子密度图直接揭 示了结合事件， 鉴定了结合的化学或者生物实体， 并且提供了配体 -干扰素复合 物的详细三维结构。 一旦命中 （hit ), 就可通过传统的筛选方法找出该命中的一 系列具有更紧密结合或期望的生物活性的类似物或衍生物。 任选地， 可将配体- 干扰素迭代暴露于额外的潜在配体，使得两次或更多次命中能优选地结合在一起 以鉴定或设计更具效力的配体。
结构同源分子的获得 /设计干扰素的模拟物
在此公开的结构坐标可用于帮助获得关于其他结晶分子或分子复合物的结 构信息。本发明的方法可以用于确定与在此公开的干 ·ί尤素具有一个或多个相似结 构特征的分子或者分子复合物的三维结构的至少一部分。 所述的分子在此称为 "结构同源的"。 相似的结构特征可包括例如氨基酸同一性区域、 保守的活性位 点或结合位点基序、 以及相似排列的二级结构元件（例如， α螺旋和 β折叠）。 在另一个实施方案中，通过比对两条氨基酸序列的残基以优化其序列长度上相同 氨基酸的个数来确定结构同源性； 在进行比对过程中，允许再这两条序列中的一 条或两条中存在空位， 以优化相同氨基酸的个数， 但是， 这些氨基酸必须在其各 自序列中保持固有顺序。 优选地， 结构同源的分子是一种具有与 SEQ ID ΝΟ:1 至少 65%同一性的氨基酸序列的蛋白质。更优选地， 与本发明的干扰素结构同源 的蛋白质包含与 SEQ ID ΝΟ:1的类似部分具有至少 80%氨基酸序列同一性的至 少 50个氨基酸残基的连续区段。 用于生成结构同源分子或分子复合物的结构信 息的方法在本领域中是公知的。
在此公开的结构坐标系还可用于解析相关干扰素、干扰素突变体或与各种配 体共复合的干扰素同系物的晶体结构。该应用使确定配体和干扰素（如候选的干 扰素改性剂和干扰素）之间相互作用的最佳位点得以可能。本方法还适用于鉴定 分子的多个结合位点中用于修饰的潜在位点。这一信息的获得为确定最有效的结 合相互作用（如在干扰素及其配体之间增强的疏水相互作用）提供了额外的途径。
此外，在一个实施方案中，本发明还提供了一种基于计算机设计重组干扰素 模拟物的方法， 包括步骤： （a )对多个模拟物生成三维结构； 和（b )将步骤（a ) 的多个模拟物的三维结构各自与包含表 7 所示的重组干扰素的原子坐标的三维 结构或该三维结构经选择的部分进行拟合，以发现所述重组干扰素的最佳拟合模 拟物， 所述原子坐标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35 埃的保守残基骨架原子优选 Ca原子的均方根偏差内变化。
合理药物设计
计算机技术可用于筛选、 鉴定、 选择和 /或设计能与干扰素或结构同源的分 子结合的化学实体或配体。在此公开的干扰素的结构坐标的信息使得具有与在此 公开的干扰素的构象互补形状的合成化合物和 /或其他分子的设计和 /或鉴定得以 可能。 具体而言， 计算机技术可用于鉴定或设计与干扰素或其部分（如 AB 或 BC环）结合的化学实体或配体， 例如受体、 改性剂、 激动剂和拮抗剂等。 潜在 的改性剂可结合干扰素的活性位点或其部分， 或干扰之， 并可以是竟争性、 非竟 争性或无竟争性的抑制剂； 或通过结合两个单体之间的界面， 从而干扰二聚化。 一旦针对生物活性进行鉴定或筛选， 这些抑制剂 /激动剂 /拮抗剂就可在治疗上或 预防上用于阻断或增强干扰素活性。还可通过计算机技术获得干扰素结合与干扰 之的配体类似物的结构 -活性数据。
当在本文中使用时， 术语 "化学实体"指化合物、 两种或更多种化合物的复 合物、及所述化合物或复合物的片段。被确定能与本发明的千扰素相结合的化学 实体就是潜在的药物候选物。 因此，如在此所鉴定的本发明的干扰素或结构同源 分子或其部分的结果的三维结构图示可有利地用于药物研发。通过本领域可获得 的多种计算机方法和技术中某一种，所述化学实体的结构坐标系可用于生成三维 图像，该三维图像又可通过计算机与所述干扰素或结构同源分子的三維图像进行 拟合。
本发明的药物设计方法的一个实施方案包括评估已知化学实体或配体与所 述干扰素或其结构同源分子的潜在结合。 因此， 所述药物设计方法还包括通过计 算机来评估所选择的化学实体或配体与本文所列的任何一种分子或分子复合物 的潜在结合。 在另一个实施方案中， 所述药物设计方法包括与本发明干扰素、 其 同系物或它们的部分结合的化学实体或配体的计算机辅助设计。化学实体或配体 可以步进方式进行设计， 一次一条片段， 或可作为整体进行设计或"从头 （de novo )，，设计。
因此，在一个实施方案中，本发明提供了一种基于计算机的合理药物设计方 法， 包括步骤： （a )提供包含表 7所示的重组干扰素的原子坐标的三维结构， 所述原子坐标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保 守残基骨架原子优选 Cot原子的均方根偏差内变化； （b )提供多个分子片段， 并对其生成三维结构； （c )将所述多个分子片段的三维结构各自与步骤（a )的 三维结构或该三维结构经选择的部分进行拟合； 和（d )将经选择的所述分子片 段组装到一个分子中以产生候选药物。在一个实施方案中， 所述方法进一步包括 步骤： （ e )获得或合成候选药物； 和（ f )令所述候选药物与所述重组干扰素接 触， 从而确定候选药物与所述重组干扰素相互作用的能力。
在本发明的某些实施方案中，所述三维结构经选择的部分包含表 7中来自第 I 25-33位氨基酸残基（ SEQ ID NO:4所示的氨基酸序列）和 /或第 44-52位氨基酸 残基（ SEQ ID NO:5所示的氨基酸序列）的一个或更多个氨基酸残基的原子坐标， 进一步地， 所述三维结构经选择的部分包含表 7 中来自第 25-33位氨基酸残基 ( SEQ ID NO:4所示的氨基酸序列）和 /或第 44-52位氨基酸残基（ SEQ ID NO:5 所示的氨基酸序列） 的原子坐标， 上述原子坐标任选地在小于约 0.65埃， 优选 ！ 约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Ca原子的均方根偏差内 变化。
同源模建
在一方面，通过同源模建，可构建或优化未被结晶的干扰素同系物的计算机 模型。首先，通过序列比对、二级结构预测、结构库 选或这些技术的任意组合， 创建出干扰素同系物的初级模型。计算软件可用于实施所述的序列比对和二级结 构预测。诸如在插入或缺少周围形成的结构碎片之类的结构不连贯可通过筛选具 有期望长度和合适构象的肽结构库从而模建获得。 若所述干扰素同系物已经结 晶， 则可通过本领域已知技术使用最终的同源模型来解析该同系物的晶体结构。 其次， 让所述初级模型能量最小化以生成能量最小化模型。该能量最小化模型可 包含违背立体化学约束的区域，在这种情况下，可使用本领域已知技术对这些区 域重新模建以获得最终的同源模型。
在另一方面，本发明提供了一种获取关于未知结构分子或分子复合物的结构 信息的方法。 在一个实施方案中， 所述方法包括以下步骤： 结晶所述分子或分子 复合物； 由所述结晶的分子或分子复合物产生 X射线衍射图； 以及将所述的 X 射线衍射图应用于在此公开的干扰素的至少一部分的结构坐标，从而产生所述未 知结构分子或分子复合物的至少一部分的三维电子密度图。
在另一方面， 本发明提供了一种干扰素同系物的模建方法。在一个实施方案 中， 所述方法包括以下步骤： 将推定的干扰素同系物的氨基酸序列与本发明的重 组干扰素的氨基酸序列进行比对并将推定的干扰素同系物的氨基酸序列掺入由 在此披露的结构坐标形成的干扰素模型中， 从而生成干扰素同系物的初级模型； 对所述初级模型进行能量最小化以生成能量最小化模型；重建所述能量最小化模 型中违背立体化学约束的区域， 从而生成干扰素同系物的最终模型。 千扰素模拟物
本发明提供了干扰素模拟物。
在一方面，本发明提供了包含在此公开的序列的肽，或其衍生物、活性部分、 类似物、 变体或模拟物， 及其用途。 因此， 在一个实施方案中， 本发明提供了一 种干扰素模拟物，其包含如 SEQ ID NO:4和 /或 SEQ ID NO:5所示的氨基酸序列。 在一个实施方案中， 将所述干扰素模拟物三维结构中的 α碳原子骨架与 IFN-a2b 三维结构中的 α碳原子骨架以最小二乘法方式进行叠合后，该干扰素模拟物在对 应于 IFNa2b蛋白质分子中第 25-33位残基上的各 a碳原子与 IFNa2b蛋白质分 子在对应位残基上的 a碳原子均方根偏差为 3.63埃 ±5%。 在某些实施方案中， 与 IFNa2b对应位残基相比， 所述干扰素模拟物在第 25-33位残基上的 a碳原子 偏差分别是 3.291埃 ±5%、4.779埃 ±5%、5.090埃 ±5%、3.588埃 ±5%、2.567埃 ±5%、 2.437埃 ±5%、 3.526埃 ±5%、 4.820埃 ±5%和 2.756埃 ±5%。 在某些实施方案中， 将所述干扰素模拟物三维结构中的 α碳原子骨架与 IFN-a2b 三维结构中的 α碳 原子骨架以最小二乘法方式进行叠合后， 该干扰素模拟物在对应于 IFNa2b蛋白 质分子中第 44-52位残基上的各 a碳原子与 IFNa2b蛋白质分子在对应位残基上 的 a碳原子均方根偏差为 2.90埃 ±5%。 在某些实施方案中， 与 IFNa2b对应位残 基相比， 所述干扰素模拟物在第 44-52位残基上的 a碳原子偏差分别是 1.614埃 ±5%, 1.383埃 ±5%、 2.735埃 ±5%、 2.709埃 ±5%、 5.018埃 ±5%、 4.140埃 ±5%、 3.809埃 ±5%、 2.970埃 ±5%和 0.881埃 ±5%。 在某些实施方案中， 所述干扰素模 拟物是功能性模拟物或结构性模拟物。在某些实施方案中， 所述干扰素模拟物是 本发明重组干扰素（rSIFN-co )的模拟物， 进一步地， 所述干扰素模拟物不包含 干复津。在某些实施方案中， 所述干扰素模拟物的三维结构类似于本发明重组干 扰素 （rSIFN-co ) 的三維结构， 尤其在相应于本发明重组干扰素的 AB环和 BC 环位置处， 两者的三维结构可以是基本上相同或完全相同的。 进一步地， 所述干 扰素模拟物的三维结构包含表 7中来自第 25-33位氨基酸残基（ AB环）和 /或第 44-52位氨基酸残基（BC环） 的原子坐标， 所述原子坐标任选地在小于约 0.65 埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Ca原子的均方 根偏差内变化。
本发明包含其中个体氨基酸可被在本领域中被认为密切相关的其他氨基酸 所取代的变体肽。 举例来说， 所述个体氛基酸可由以下方式进行取代： 可用任何 疏水性脂肪族氨基酸取代其他疏水性脂肪族氨基酸；可用任何疏水性芳香族氨基 酸取代其他疏水性芳香族氨基酸；可用任何带有极性侧链的中性氨基酸取代其他 带有极性侧链的中性氨基酸； 可用任何酸性氨基酸取代其他酸性氨基酸； 可用任 何碱性氨基酸取代其他碱性氨基酸。 当在本文中使用时， "模拟物"、 "功能性 / 结构性模拟物" 均指具有与在此公开的多肽相同的功能 /结构活性的肽变体或有 机化合物。这样的模拟物或者类似物的实例包括： 被模建以比拟在此公开的干扰 素的三维结构 （该三维结构包含表 7所示的重组千扰素的原子坐标）， 特别是如 上所述的氨基酸残基的排列的化合物或肽。 因此， 当在本文中使用时， "本发明 重组干扰素的模拟物" 指与具有与本发明重组干扰素 （ rSIFN-co )相同的功能 / 结构活性但不同于本发明重组干扰素的肽变体或有机化合物，尤其是具有与本发 明重组干扰素的 AB环和 /或 BC环相同的空间结构但不同于本发明重组干扰素的 肽变体或有机化合物。 当作为肽变体时， 这样的 "模拟物" 的氨基酸序列一般具 有与本发明重组千扰素的氨基酸序列类似的长度，例如可包含约 120-200个氨基 酸残基， 优选约 140-180个氨基酸残基， 更优选约 150-175个氨基酸残基， 还更 优选约 160-170个氨基酸残基， 例如约 164、 165、 166或 167个氨基酸残基； 或 者， 这样的 "模拟物" 可以是包含本发明重组干扰素的 AB环和 /或 BC环的具有 较短氨基酸序列长度的肽变体， 例如其可包含约 10-100个氨基酸残基， 优选约 15-80个氨基酸残基。
可通过本领域通常所知的模建技术生成适合的模拟物或者类似物。包括设计 涉及研究功能相互作用的 "模拟物"以及设计包含以使之能产生上述相互作用的 方式排列的官能团的化合物。
对已知药学活性化合物设计模拟物是一种基于 "前导"化合物用于开发药物 的已知方法。这一方法可用于获得不易合成或需高价合成的活性化合物， 也可用 于改良不宜于常规给药的活性化合物； 所述不宜于常规给药的活性化合物包括： 会在消化道中被蛋白酶快速分解，从而不宜于口服的活性化合物多肽。模拟物设 计、 合成和测试可用于避免针对目标性质随机筛选大量分子。
在根据具有给定目标特性的化合物 /肽设计模拟物的过程中， 通常包括以下 几步： 首先， 确定对于决定目标性质起关键和 /或重要作用的化合物 /肽的特定部 分。 就肽而言， 可通过系统性地改变肽中的氨基酸残基从而实现这样的确定， 例 如将每个残基依次取代。 构成化合物活性区的这些部分或残基被称为其 "药效 团'，。
一旦发现所述药效团， 就可根据其物理性质对其结构进行模建； 所述物理性 质例如立体化学性、 键合、 大小和 /或电荷、 该模建使用了来自多种来源例如光 谱技术、 X-射线衍射和 NMR的数据。 计算分析、 相似性制图 （其模拟了药效团 的电荷和 /或体积， 而非原子间键合） 以及其他技术可用于该模建过程中。 在该 方法的变型中，模建配体及其结合伴侣的三维结构。这一方法特别适用于配体和 /或结合伴侣在结合中产生构象变化的情况， 从而容许在模拟物设计中对该模型 作进一步考虑。 然后， 选择可将模拟药效团的化学基团移植到其上的模板分子。可便利地选 择所述模板分子及移植到其上的化学基团，从而容易地合成所述模拟物； 而所合 成的模拟物除了保持前导化合物的生物活性外，还可能是药理学可接受的， 并且 不会在体内降解。通过这一手段获取的模拟物经筛选后， 即可得知其是否具有目 标性质或者在什么程度上显示了目标性质。 然后， 可进行进一步的优化和改性， 以获得用于体内或临床测试的一种或多种最终的模拟物。
在另一方面， 本发明还提供了一种非配体化的分子； 所述分子具有在此公开 的干扰素的至少一部分。 例如， 所述非配体化结合的分子可包含 SEQ ID NO: 4 或 SEQ ID NO: 5 所示序列 （分别为在此公开的干扰素的 AB环和 BC环的序 歹' J ), 进一步地所述非配体化的分子具有如 SEQ ID NO: 4 或 SEQ ID NO: 5 所 示序列。 组合物及治疗应用
本发明提供了包含本发明的重组干扰素晶体或本发明的重组干扰素模拟物 的组合物。 在一个实施方案中， 所述组合物是药物组合物。 在一个实施方案中， 所述药物组合物进一步包含有药学上可接受的载体。
无论是就本发明而言用于人体给药的多肽、 抗体、 肽、 核酸分子、 小分子、 模拟物还是其他药学上有用的化合物， 优选的给药剂量都是 "预防有效剂量"或 "治疗有效剂量"（当然预防也可被认为是治疗的一个方面），这样的剂量足以显 示对个体的有益效果。本发明的实际给药剂量、给药频率以及给药时间随受治疗 疾病的性质和严重程度而定。 治疗处方， 如剂量确定等问题， 由医生或其他医务 工作者按情况而定。 药物组合物可依据情况单独给药或联合给药。
根据本发明并供本发明所用的药物组合物， 除活性成分外， 可包括： 药学上 可接受的赋形剂、 载体、 緩冲剂、 稳定剂或其它本领域公知的物质。 这类物质应 当是无毒且不会影响活性成分效力的物质。载体或其他物质的确切性质依据给药 方式而定； 所述给药方式包括口服和注射； 而注射则进一步包括皮下注射、 肌肉 注射以及静脉注射等。 上述技术和给药方案的实例可见 Remington's Pharmaceutical Sciences, 第 16版， Osol, A. (编）， 1980。
在某些实施方案中， 所述药物组合物可配制成如下剂型， 包括： 片剂、胶囊、 口月^液、 贴剂、 注射剂、 喷雾剂、 栓剂、 溶液制剂， 推荐的剂型为注射剂。 可皮 下或静脉注射给药，药物组合物中的载体可使用任何一种适宜的可接受的药物载 体， 包括粘合剂、 崩解剂、 润滑剂、 填充剂、 增溶剂、 緩沖剂、 防腐剂、 增稠剂、 配合剂和其他佐剂。
根据本发明的不同目的， "药学上可接受的载体"是指任何标准的药用载体。 例如公知的适当载体包括但不仅限于任何标准药用的载体如：磷酸盐緩冲液及各 种润湿剂。 其它载体可能包括用于片剂、 颗粒剂及胶嚢等的添加剂。 典型的载体 常含有如： 淀粉、 乳液、糖、 纤维素、 某种类型的粘土、 明胶、硬脂酸或其盐类、 硬脂酸镁或硬脂酸钙、 滑石、 植物油脂、 树胶、 乙二醇或其它已知的赋形剂。 这 些载体中也可能还含有调味剂及增色剂及其它成分。这些载体中的成分可用公知 的传统方法调制。
进一步地， 由于本发明重组干扰素的模拟物具有本发明重组千扰素的 AB环 和 /或 BC环结构 （如上述特定的 AB环和 /或 BC环空间结构）， 因此预计其能与 本发明的重组千扰素一样， 用于治疗病毒性疾病和 /或肿瘤。
因此， 在另一方面， 本发明提供了本发明的重组干扰素晶体、 干扰素模拟物 或包含上述晶体或模拟物的组合物在制备用于治疗病毒性疾病和 /或肿瘤的药物 中的应用。
在另一方面， 本发明提供了一种治疗病毒性疾病和 /或肿瘤的方法， 所述方 法包括向受试者施用有效量的本发明的重组干扰素晶体、干扰素模拟物或包含上 I 述晶体或模拟物的组合物。
在另一方面， 本发明还提供了一种用于治疗病毒性疾病和 /或肿瘤的药物组 合物， 所述药物组合物包含有效量的本发明的重组干扰素晶体、干扰素模拟物或 包含上述晶体或模拟物的组合物。
在某些实施方案中， 所述病毒性疾病可包括： 曱型肝炎、 乙型肝炎、 丙型肝 i 炎、 其它类型的肝炎、 由 Epstein-Barr病毒、 人体免疫缺陷病毒（ HIV ) 、 埃博 拉病毒、 严重急性呼吸道综合征病毒（SARS ) 、 流感病毒、 巨细胞病毒、 单纯 疱疹病毒、 或其它类型的疱疹病毒、 乳头多瘤空泡病毒、 痘病毒、 小核糖核酸病 毒、 腺病毒、 鼻病毒、 I型人类 T细胞白血病病毒、 或者 II型人类 T细胞白血病 病毒， 或者 III型人类 T细胞白血病病毒引起的病毒感染。
) 在某些实施方案中， 所述肿瘤是癌症、 实体瘤， 所述肿瘤可包括： 皮肤癌、 基底细胞癌及恶性黑色素瘤、 肾细胞癌、 肝癌、 曱状腺癌、 鼻咽癌、 实体肿瘤、 前列腺癌、 胃 /腹癌、 食道癌、 直肠癌、 胰癌、 乳腺癌、 卵巢癌、 浅表膀胱癌、 血管瘤、表皮样癌、 子宫颈癌、 非小细胞肺癌、 小细胞肺癌、神经胶质瘤、血癌、 急性血癌、 慢性血癌、 慢性骨髓性白血病、 毛细胞白血病、 淋巴腺瘤、 多发性骨 髓瘤、 红血球过多病、 卡波济氏肉瘤。
本发明将通过下列实施例得以详细描述， 所述实施例仅作为解释说明目的， 并不意在限制本发明的范围。 可对本发明进行修改而不背离的本发明的范围。
所有出版物、 专利和专利申请在此以其整体并入作为参考， 如同每篇独立出 版物、 专利或专利申请被明确且单独地指出以其整体并入作为参考一样。 具体实施方式
实施例 1 重组千扰素 rSIFN-co的制备 本实施例为重组干扰素 rSIFN-co (原液）的制备方法（具体参见美国专利第 7,364,724 号说明书实施例 1和 2以及中国专利公开号 CN1740197A说明书中第 11-17页记载的方法）。
一、 基因克隆：
根据已发表的干复津的编码 DNA序列及推断的氨基酸序列资料（ Klein ML等， Structural characterization of recombinant consensus interferon- alpha. Journal of Chromatography, 1988, 454: 205- 215 ), 利用大肠杆菌优先表达密码子 （ The Wisconsin Package, by Genetics Computer Group, Inc. Copyright 1992, Medison, Wisconsin, USA ) , 在保证氨基酸序列不变的情况下， 对其 DNA编码序列进 行分子设计， 然后人工合成 rSIFN-co全长 cDNA编码基因。
合成 rSIFN-co cDNA序列
rSIFN-co cDNA 5'-端和 3'-端半分子的合成
用 PCR方法直接合成 rSIFN-co cDNA 5'-端 280bp ( I 片段） 和 3'-端 268bp( II片段）两个 cDNA半分子。片段 I的 3'-端与片段 II的 5'-端有 41bp 的核苷酸序列重叠互补。 化学合成如下寡聚脱氧核苷酸片段
Oligomer A：
TGTGCGACC
GCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGAC3'
Oligomer B:
Oligomer C:
： TGCTGGTA
AGGAGTCTTTGGTGGAGAACAGGTTGAAGGTCTGCTGGATCATTTC3'
Oligomer D:
，CCCTGCTG'
TTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAAC3'
Oligomer E:
\AGAAACC
CGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGG3'
Oligomer F:
( 2 ) PCR反应
PCR反应 I合成 rSIFN- co 5'-端半分子： 用寡聚脱氧核苷酸片段 B作 为模板， A和 C 两个寡聚脱氧核苷酸片段作为引物， 进行 PCR反应合成 长度为 280bp的 rSIFN-co 5'-端半分子产物。
PCR I 反应混合物如下： （单位： μ 1 ) (总体积 :50 μ 1 ) 无核酸酶消毒蒸馏水 39
10 X Pfu反应緩沖液（美国 Stratagen公司生产） 5 dNTP混合物 （每一 dNTP浓度为 2.5mmol/L ) 2
A片段引物 （25 μ πιο1/υ 1
C片段引物 （25 μ πιο1/υ 1 B片段模板 ( 1 μ mol L ) 1 Pfu DNA聚合酶（美国 Stratagen公司生产）（ 25U/ μ 1 ) 1 PCR I反应周期为：
95 °C 2'→ ( 95 °C 45"→65°C l'→72°C 1 ' ) > 25周期→72°C 10'→4°C PCR反应 II合成 rSIFN-co 3'-端半分子： 用寡聚脱氧核苷酸片段 Ε作 为模板， D和 F两个寡聚脱氧核苷酸片段作为引物， 进行 PCR反应合成长 度为 268bp的 rSIFN-co 3'-端半分子产物。
PCR II反应混合物如下： （单位： μ 1 ) (总体积 :50 μ 1 ) 无核酸酶消毒蒸饱水 39
10 X Pfu反应緩冲液（美国 Stratagen公司生产） 5
dNTP混合物 （每一 dNTP浓度为 2.5mmol/L ) 2
D片段引物 （ 25 μ mol/L ) 1
E片段引物 （ 25 μ mol/L ) 1
F片段模板 ( 1 μ mol/L ) 1
Pfu DNA聚合酶（美国 Stratagen公司生产）（ 25U/ μ 1 ) 1
PCR II反应条件和周期同 PCR I。
rSIFN-co全长 cDNA分子的组装
采用 "重叠 -延伸 PCR" 方法将上述 PCR合成的 I和 II片段组装在一 起从而得到完整的 rSIFN-co cDNA全长分子序列。 并且在其 5'-端和 3'-端 分别引入 Nde I和 Pst I限制性酶切位点， 以便于将 rSIFN-co cDNA序列 克隆到质粒载体中去。
( 1 ) 化学合成引物
Oligomer G: 5'ATCGGCCATATGTGCGACCTGCCGCAGACCC3'
Oligomer H: 5'ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3' ( 2 ) "重叠 -延伸 PCR" 反应
PCR 反应混合物： （单位： μ 1 ) (总体积 :50 μ 1 ) 无核酸酶消毒蒸榴水 38
10 X Pfu反应緩冲液（美国 Stratagen公司生产） 5
dNTP混合物 （每一 dNTP浓度为 2.5mmol/L ) 2 引物 G ( 25 μ mol/L ) 1
引物 H ( 25 M mol/L ) 1
*片段 I PCR产物 （ 1 μ mol/L ) 1
*片段 II PCR产物 （ 1 μ πιοΙ/L ) 1
Pfu DNA聚合酶（美国 Stratagen公司生产）（ 25U/ μ 1 ) 1
注： 用美国 Stratagen公司生产的 StrataPrep PCR纯化试剂盒将 PCR产物先进行 分离纯化， 然后溶于消毒蒸馏水中。
PCR反应条件及周期同前述 PCR I 。
rSIFN- co基因的克隆及序列分析
选用 pLac T7质粒作为 rSIFN-co cDNA基因克隆的载体。 pLac T7质 粒是经 pBluescript ll KS ( + ) 质粒 （美国 Stratagen公司生产） 改造而成。
用 StrataPrep PCR纯化试剂盒（美国 Stratagen公司生产）将含 rSIFN-co 全长 cDNA PCR产物进行纯化，然后用 Ndel和 Pstl进行酶切；同时将 pLac T7质粒进行 Ndel和 Pstl双重酶切。 将这二种酶切 DNA片段在 1%琼脂糖 胶上进行电泳分离，然后用美国 Promoga公司的 Winzard DNA纯化试剂盒 从胶中回收，纯化 507bp长的 rSIFN-co DNA片段和 2.9kb的质粒酶切 DNA 片段。 将二者经 T4 DNA连接酶催化连接成重组质粒。 将连接反应混合物 转化 DH5 α感受态细胞 （美国 Gibco公司生产）。 经 37 °C过夜培养后， 挑 选阳性重组菌落， 命名为 pHY-l。
按 DNA序列分折试剂盒（ SequiThermTM Cycle Sequencing Kit, 购自 美国 Epicentre Technologies公司 ） 说明书进行 DNA序列测定反应， 其中 引物为通用 T7和 T3引物， DNA测序结果显示其与理论设计相符。
纯化的重组 rSIFN-co蛋白进行 N-末端 16个氨基酸及 C-末端 4个氨基 酸序列测定。 其结果为：
N端：
Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn-Arg-Arg-Ala-Leu- 其 N-末端的曱硫氨酸（ Met ) 在成熟蛋白中被切除。
C端：
Arg-Arg-Lys-Glu-COOH。 rSIFN-co全长核苷酸序列如 SEQ ID NO:2所示， 氨基酸序列如 SEQ ID NO:l所示。
表达载体的构建、 转化、 酶切鉴定及其遗传稳定性
表达载体的构建、 转化
将大肠杆菌表达载体 pBAD18 质粒先经 Nde l酶解， 使质粒线性化 ( Linearized), 然后用 Xba I进行充分酶解。 经 1<¾琼脂糖凝胶电泳， 再用 德国 QIAGEN公司生产的 QIAEXII试剂盒纯化， 得到 pBAD18 经 Ndel 和 Xbal消化的 4.8kb片段。
与此同时将 pHY-1质粒进行 Nde I和 Xba I双酶切， 同样经 1%琼脂 糖凝胶电泳分离后， 纯化出 715bp 大小的序列片段。 将上述 4.8kb 的 pBAD18片段与 715bp的 rSIFN- co和 pBAD18酶切片段在 T4 DNA连接酶 催化下连接成重组质粒。 将连接反应物转化 DH5a感受态细胞， 然后将转 化细胞涂于 LB-Amp琼脂平板， 置 37°C培养过夜。
阳性克隆菌林的筛选
随机从上述 LB 平板中挑起单个细菌菌落， 用核酸内切酶酶解， PCR 分析的方法筛选含 rSIFN-co 全长编码序列的重组质粒菌株。 将其中一个 PCR阳性重组质粒命名为 pHY-5。将含有 pHY-5质粒的菌株命名为 PVIII , 扩增后加入甘油冻存液冻存于 - 80 'C备用。
rSIFN-co基因在 E.coli LMG194中的高效表达
在 pHY-5质粒中， rSIFN-co基因处于强启动子 PBAD的调控之中， 而 PBAD又受 AraC蛋白的调控。 AraC是由位于同一质粒中的 araC基因编码 的蛋白质。 在没有阿拉伯糖存在的情况下， AraC二聚体与 02及 12结合形 成一个 210bp的环。 这一结构导致转录的完全抑制。 当加入阿拉伯糖时， AraC二聚体与 02脱离， 并转而与 Ii和 12结合， 解除对转录的抑制。 阿拉 伯糖结合失活、 抑制、 及激活 PBAD启动子的转录， 从而刺激 PBAD介导高 水平的 rSIFN-co表达。 rSIFN-co表达量可达菌体总蛋白的 50%。 二、 分离纯化：
( 1 )、 制备生产菌种： 将含有表达载体 pHY-5的 E. coli LMG194工程菌种 接种在 LB培养基中， 37。C , 摇瓶振荡 （200 rpm )培养过夜（约 18小时）， 细 菌培养液中加入 50 %>浓度为 30 %的甘油， 混勾分装成 1 ml每支， -20°C保存， 作为生产菌种；
( 2 )、 制备 I级种子菌： 将生产菌种按 1 «¾的比例接入 LB培养基（ 1 L中含 有胰蛋白胨 10 g, 酵母膏 5 g， 氯化钠 10 g ), 37°C , 200rpm培养过夜， 作为 I 级种子菌；
( 3 )、 发酵、 收菌： 将 I级种子菌再按 10 %的比例加入 RM培养基（1 L中 含有酪蛋白 20 g, 氯化镁 1 mmol/L ( 0.203 g ),磷酸氢二钠 4 g,磷酸二氢钾 3 g， 氯化钠 0.5 g, 氯化胺 1 g )中， 37°C , pH 7.0, 发酵至 00600值 2.0左右加入阿拉 伯糖（20 % )至终浓度 0.02%诱导， 4小时后收菌， 离心， 得到菌体沉淀；
( 4 )、 制备包涵体： 将离心后的菌体沉淀用适量緩冲液 A ( 100 mmol/L Tris 盐酸 pH 7.5, 10 mmol/L EDTA, 100 mmol/L氯化钠）重混悬，置- 20°C冷冻过夜， 取出融化后， 匀浆机破菌， 离心， 再用緩冲液 B ( 50 mmol/L Tris盐酸 pH 7.5，
1 mol/L尿素， 10 mmol/L EDTA, 0.5% Triton X-100 )、緩冲液 C ( 50 mmol/L Tris 盐酸、 pH 7.5， 2 mol/L尿素， 10 mmol/L EDTA, 0.5% Triton X-100 )分别洗涤 沉淀两次， 再用蒸媳水洗涤一次， 得到包涵体；
( 5 )、 复性处理： 用 6 mol/L盐酸胍 (或尿素）溶解包涵体得到稍浑浊的变 性液， 10000 rpm高速离心，取上清液测定变性液的蛋白浓度； 按终蛋白浓度 0.3 mg/ml, 分三次将变性液加入已配制好的复性液（0.5 mol L精氨酸， 150 mmol/L Tris盐酸 pH 7.5, 0.2 mmol/L EDTA ) 中， 并在 4°C连续搅拌过夜（约 18 小时）； 然后， 依次用 10倍体积的 10 mol/L磷酸盐緩冲液（PB )、 和 5 mol/L的嶙酸盐 緩冲液及蒸馏水透析； 透析完毕， 用 2 mol/L的醋酸 -醋酸钠 （ pH 5.0 )调 pH, 静置， 过滤；
( 6 )、 HS阳离子柱层析： 柱体先用 20 mmol/L的醋酸 -醋酸钠 （ pH 5.0 ) 平衡， 将步骤（ 5 )得到的复性产物以 30 ml/min的速度上样， 用 20个柱体积的 20 mmol/L醋酸 -醋酸钠（ pH 5.0 )，洗脱杂蛋白；接着，用 5个柱体积含 0.15 mol L 氯化钠的 20 mmol/L醋酸-醋酸钠 （pH 5.0 ), 洗脱杂蛋白。 然后， 再用 3个柱 体积含 0.18 mol L氯化钠的 20 mmol/L醋酸 -醋酸钠 （ pH 5.0 ), 洗脱杂蛋白； 最后， 用含 0.25 mol/L氯化钠的 20mmol L醋酸-醋酸钠 （ pH 5.0 )解离目标蛋 白， 得到 HS解离蛋白液；
( 7 )、 铜离子亲和层析（ chelating sepharose™ fast flow ): 将 HS解离蛋白液 加入 0.2 mol/L, pH 6.6的 PB緩冲液中， 用 4 mol/L的氯化钠调到含 1 mol L氯 化钠， pH 6.0, 备上样； 柱体用含 1 mol L氯化钠的 50 mmol/L碑酸氢二钠緩冲 液（pH 5.5 )平衡， 以 1 ml/min的速度上样； 接着， 用 50 mmol/L磷酸氢二钠緩 冲液（pH 5.0 )， 洗脱杂蛋白； 再用 50 mmol/L磷酸氢二钠緩冲液（pH 4.0 ), 洗 脱杂蛋白； 然后， 再用 50 mmol/L嶙酸氢二钠緩冲液（pH 3.6 ), 解离目标蛋白， 得到螯合柱解离目标蛋白液；
( 8 )、 HS柱层析： 将螯合柱解离目标蛋白液稀释 30倍， 并将 pH调至 5.0， 上 HS柱， 以含 0.5 mol/L氯化钠的 PB緩冲液（ pH 7.0 )解离， 收集， 即得所述 的重组干扰素 （原液）。 实施例 2 重组干扰素制剂 冻干注射剂 （冻干 的配方
本发明 rSIFN-co原液 34.5 g/ml
pH 7.0的碑酸盐緩冲液 10mmol/L
甘氨酸 0.4mol/L 制备工艺： 按配方称料， 无菌无热原注射水溶解， 0.22μιη 孔径滤膜过滤除 菌， 于 6-1CTC保存， 取样作无菌和热原检查合格后分装西林瓶中， 每瓶 0.3-0.5 的单次剂量， 分装后放置至冻干机中冷冻干燥。
水溶液注射剂的配方
本发明 rSIFN-co原液 34.5 g/ml
pH 7.0的磷酸盐緩冲液 25mmol/L
氯化钠 0.4mol/L 制备工艺： 按配方称料， 无菌无热原注射水溶解， 0.22μιη孔径滤膜过 滤除菌， 于 6-10Ό保存，取样作无菌和热原检查合格后分装于密闭容器中， 每瓶 0.3-0.5的单次剂量， 分装后成品置 2-10Ό暗处保存。 实施例 3 rSIFN-co与千复津抗人乳腺癌细胞的体外实验研究 本实施例为重组干扰素 rSIFN-co及干复津抗人乳腺癌细胞的体外实验研究。 分别以本发明所述重组干扰素 rSIFN-co和美国安进公司生产的干复津为试 验药物， 考察其对人乳腺癌 MCF-7细胞及耐药株 MCF-7/ADR细胞增殖、 凋亡 及癌基因表达的影响。
一、 实睑方法
1. 细胞培养
将人乳腺癌细胞株 MCF-7细胞及人乳腺癌阿霉素耐药株 MCF-7/ADR细胞 分别培养于 25cm2或 75cm2培养瓶中，细胞长满瓶底后，以 0.25%胰酶消化传代， 取对数生长期细胞进行实验。
2. MTT比色法检测不同浓度的 rSIFN-co对细胞增殖的影响
实验分组: 每株细胞分 3大组共 11小组: rSIFNco组（0.02、 0.078、 0.313、 1.25、 5.(^g/ml )、 干复津组（0.02、 0.078、 0.313、 1.25、 5.(^g/ml )、 空白对照组 (含 10%小牛血清的 RPMI1640培养液（美国 Sigma公司）， 也称为 RPMI1640 完全培养基）， 其中用 RPMI1640完全培养基将 rSIFNco和干复津稀释成所需浓 度（使乙醇浓度 <1% ), 4°C保存。
取对数生长期的 MCF-7 细胞、 MCF27/ADR 细胞， 用含 10%小牛血清的 RPMI1640培养液，配制成 1.25xl05/mL的细胞悬液，台盼蓝法测细胞活率 > 95%, 将细胞接种于 96孔培养板上， 每孔 100μί。 按上述分组加入药物后 24h、 48h、 72h, 常规 MTT法检测细胞的增殖（在酶标仪上于波长 490nm处检测吸光度）。 每组设 2个复孔作为平行样。 以上实验重复 3次，按照以下公式计算不同药物浓 度及作用时间对细胞株的生长抑制率：
增殖抑制率 （<¾ ) = (对照组 A值-实验组 A值） /对照组 A值 χ100%。
3. 流式细胞仪 ( FCM )检测细胞凋亡 实验分组：每株细胞分 3组，分别为 rSIFN-co( 5 g/mL )组、干复津（ 5 g/mL ) 组、 及空白对照组（含 10%小牛血清的 RPMI1640培养液）。
FCM检测： 分别收集加入药物作用 48h后各组细胞，制成单细胞悬液， 采用 碘化丙啶（PI )染料， 用 Elite Esp型流式细胞仪（美国 Coulter公司）测定凋亡 率， 用随机所附软件对测量值进行分析。 以上实验重复 3 次。
4. 免疫组化检测各组细胞癌基因的蛋白表达
实验分组： 每株细胞分 3 组。 向 MCF- 7 细胞培养液中加入 rSIFN-co ( 5 g/mL )、 干复津（5 g/mL )、及含 10%小牛血清的 RPMI1640。 MCF-7/ADR 细胞培养液中加入 rSIFN-co ( 5 g/mL ) 、 干复津（5 g/mL ) 、 及含 10%小牛血 清的 RPMI1640。
免疫组化检测 P53、 Bcl-2、 CerbB-2蛋白的表达： 将细胞爬片所用的盖玻片 先经酸处理后洗涤干净、高压灭菌，放入 6 孔培养板中。取对数期生长的 MCF27 细胞、 MCF27/ADR 细胞， 用 0.25%胰酶消化成单细胞悬液， 接种至 6孔板中， 每孔 lxlO5, 37。C、 〇02孵箱培养 24h, 待细胞贴壁后， 分别加入各组药物， 处 理 48 h后，取出盖玻片，采用常规免疫组化 SABC法染色，一抗浓度均为 1:100。
结果判定标准：染色结果判定参照 Volm的方法（ Volm M等， European Journal of Cancer, 1997， 33(3):691-693 ),以细胞胞核（ P53 )、胞浆（ Bel- 2 )或胞膜（ CerbB-2 ) 内出现黄色或棕黄色颗粒者为阳性。 高倍镜下 （ 400x )对每张玻片随机选取 5 个视野， 每个视野计数 200个细胞。 每组细胞的表达由两方面共同决定。 阳性染 色深度: 按每个细胞着色深浅计分， 无着色计 0分， 浅黄色计 1分， 棕黄色计 2 分， 椋褐色计 3分， 其平均值即为该组细胞的平均染色深度； 阳性细胞百分比： 未见染色为 0分， 染色细胞< 25%为 1分， 25 % ~ 50 %为 2分， > 50%为 3分。 两项的总和， 得分 0〜分判为表达阴性， 2 ~分判为阳性， 4 ~ 6分判为强阳性。 使用双盲法进行（染色者及结果观察者均不知玻片的分组情况） 。
二、 统计学方法
对试验数据进行统计学处理：所有试验数据，经 SPSS 11.5统计软件包处理， 采用 t检验、 方差分析和秩相关分析； < 0.05, 认为差异有统计学意义。
三、 实验结果
1. 对 MCF-7细胞、 MCF-7/ADR细胞增殖的影响 ( 1 ) MCF-7细胞： rSIFN-co能抑制 MCF-7细胞的增殖。 浓度分别为 0.02、 0.078、 0.313、 1.25、 5.(^g/mL 的 rSIFN-co 和干复津作用的各细胞组的吸光度 OA, 与空白对照组比较均显著降低， 经方差分析， 早期 （24h、 48h ) rSIFN-co 与干复津对 MCF-7细胞抑制作用的差异无统计学意义（尸>0.05 ) , 但作用 72h 后， 除最低浓度 0.02 g/mL外， rSIFN-co的抑制率均较相同浓度干复津的抑制 率更高， 差异有统计学意义（Z^O.05 ) 。 见表 1-1。
对 MCF-7细胞体外生长的抑制率（％ , n=6 )
Figure imgf000032_0001
* P < 0.05 , vs干复津
( 2 ) MCF-7/ADR细胞： rSIFN-co能抑制 MCF-7/ADR细胞的增殖。 浓度分 别为 0.02、 0.078、 0.313、 1.25、 5.(^g/mL的 rSIFN-co和干复津作用的各细胞组 的吸光度 OA, 与空白对照组比较均显著降低， 经方差分析， 除最低浓度 0.02 g/mL外， rSIFN-co的抑制率均较相同浓度干复津的抑制率更高， 差异有统计学 意义（尸 <0.05 ) 。 见表 1-2。
表 1-2 对 MCF-7/ADR细胞体外生长的抑制率（％ , n=6 )
Figure imgf000032_0002
* < 0.05, vs干复津
2. 对 MCF-7细胞、 MCF-7/ADR细胞凋亡的影响
与空白对照组相比， 作用 48h后， 5 g/mL的 rSEFN-co和干复津均能诱导 MCF-7 细胞凋亡， 与对照组相比， 差异有统计学意义（P<0.01 ) , 5 g/mL 的 rSIFN- co和干复津均能诱导 MCF-7/ADR细胞凋亡， 与对照组相比， 差异有统计 学意义（Ρ<0.01 )。 相同浓度的 rSIFN-co较干复津对 MCF-7细胞、 MCF-7/ADR 细胞的诱导凋亡作用更显著， 两者差异有统计学意义（尸<0.01 ) 。 见表 1-3。
表 1-3 作用 48h时乳腺癌细胞凋亡率的变化 ( % , n=6 )
Figure imgf000033_0001
* P < 0.01 , vs对照； A/^ O-Ol, vs干复津
3.对各组细胞 P53、 CerbB-2和 Bel- 2蛋白表达的影响
在 MCF-7细胞中， rSIFN-co处理后能下调 P53表达， 与对照组相比， 差异 有统计学意义（P<0.05 ), 但干复津处理后， 虽然 P53表达有一定程度下降， 但 与对照组相比， 差异无统计学意义（P>0.05 ); 在 MCF-7/ADR细胞中， rSIFN- co 和干复津均能下调 P53表达， 与对照组相比， 差异有统计学意义（尸<0.05 ), 但 相同浓度的 rSIFN-co与干复津相比， 两者间无明显差异 （Ρ>0.05 )。
在 MCF-7和 MCF-7/ADR细胞中， rSIFN-co处理后均能下调 CerbB- 2表达， 与对照组相比， 差异有统计学意义（尸<0.01 ), 干复津处理后， 虽然 CerbB- 2表 达均有一定程度下降， 但与对照组相比， 差异无统计学意义（尸〉 0.05 )。
在 MCF-7细胞中， rSIFN-co和干复津处理均能上调 Bcl-2表达， 与对照组 相比， 差异有统计学意义（尸 <0.01 ), 且 rSIFN-co上调 Bcl-2表达的作用较干复 津更显著， 两者差异有统计学意义（ P=0.001 )；在 MCF-7/ADR细胞中， rSIFN-co 和干复津处理均能上调 Bcl-2表达，与对照组相比，差异有统计学意义（Ρ<0.01 )。
结果详见表 1-4。
表 1-4 作用 48h后对乳腺癌细胞 P53、 CerbB-2, Bcl-2表达的影响 （ n=5 )
Figure imgf000033_0002
Figure imgf000034_0001
* F < 0.05 , * P < 0.01 , vs对照 实施例 4
rSIFN-co与千复津抗宫颈癌细胞的体外实验研究 本实施例为重组干扰素 rSIFN-co及干复津对宫颈癌细胞的生长抑制及促凋 亡作用的体外实验研究。
分别以本发明所述重组干扰素 rSIFN-co和美国安进公司生产的干复津为试 验药物， 观察其对宫颈癌 Caski 细胞（HPV16 + )细胞的生长抑制以及促凋亡作 用。
一、 实验方法
1、 Caski细胞生长抑制试验
1.1 细胞培养与分组
用含 10 %小牛血清的 RPMI-1640培养液稀释药品， 在 96孔培养板中培养 宫颈癌 Caski细胞。 以培养液调定细胞数为 1 X 105/ml制成单细胞悬液， 在培养 板各孔中加入 ΙΟΟμΙ细胞悬液。向培养板中分别加入 rSIFN-co和干复津，每种药 物的浓度梯度为 0.1564g/ml、 0.625 g/ml、 2.5 g/ml和 lC^g/ml,并设置对照组（含 10 %小牛血清的 RPMI-1640培养液）， 每个浓度设 3 个复孔。放置于 37°C、 5% C02孵箱培养 72小时。
1.2 MTT法检测细胞生长抑制率
配制 MTT试剂 （美国 Sigma公司）， 使其浓度为 5mg/ml。 培养板各孔分别 加入 ΙΟμΙ MTT液， 轻微震荡使其混匀， 放置于 37°C， 5% C02孵箱中反应 4小 时后， 见培养板孔底部出现蓝色结晶。 弃去培养板中上清液， 每孔加入 ΙΟΟμΙ二 曱基亚砜， 室温下待结晶溶解， 在 570nm波长酶标仪读取 OD值。
1.3 计算细胞生长抑制率
细胞生长抑制率 =(1-样品孔 OD值 /对照孔 OD值） X 100%。
2、 Caski细月包凋亡试-睑
2.1 细胞培养与分组 分 Ί组在含 10 %灭活小牛血清的 RPMI-1640培养液中培养宫颈癌 Caski细 胞， 第 1组直接用培养液培养 72 小时， 作为对照组； 第 2-4组 rSIFN-co浓度分 别为 0.156 g/ml、 0.625 g/ml、 2.5 g/ml; 第 5-7组干复津浓度分别为 0.156 g/ml、 0.625 g/ml、 2 g/ml。
2.2 流式细胞仪检测 Caski细胞凋亡率
收集每组细胞至离心管中以 1000r/min 离心 5 分钟， 弃去上清液， 采用 AnnexinV/PI双染色法检测细胞凋亡。 每样本细胞数约为 1 x 106个， 用孵育緩冲 液洗 1次， 1000r/min离心 5分钟， 用 ΙΟΟμΙ的标记液重悬细胞， 室温下避光孵 育 15分钟， 以 1000r/min离心 5分钟沉淀细胞， 孵育緩冲液洗 1次， 加入荧光 溶液在 4°C下孵育 20分钟， 避光并不时震动， 以流式细胞仪检测。
二、 统计学处理
所有定量分析数据以 JT±S表示，采用方差分析不同药物、 不同浓度组之间的 差异,应用 SPSS 14.0软件包进行统计学分析。
三、 实验结果
1、 对 Caski细胞生长抑制的作用
rSIFN-co和干复津对 Caski细胞的生长有抑制作用， 这种作用随干扰素作用 浓度的增加而升高。 rSIFN-co与干复津相比， 在 0.625、 2.5、 lO g/ml浓度时对 Caski细胞生长的抑制作用高于千复津， 两者差异有统计学意义（Ρ< 0.01)。 见 表 2-1。
对宫颈癌细胞的抑制作用 （JF±S)
细胞生长抑制率
药物浓度 ( \g /ml )
rSIFN-co 干复津
0.156 8.6 ±2.1 7.3±2.2
0.625 17.6 ±3.3① 7.4 + 4.0
2.5 42.7 ± 1.5① 9.7 + 1.6
10 67.3 ±4.4① 53.0 ±5.8
①与同浓度干复津组比较， /^O.OL
2、 促进 Caski细胞凋亡的作用
rSIFN-co和干复津都有促进宫颈癌细胞凋亡的作用，这种作用随药物浓度的 增加而明显； rSIFN-co在低浓度 ( 0.156 g /ml )时对宫颈癌细胞的促凋亡作用强 于干复津的作用， 两者差异有统计学意义（Ρ< 0.01) 。 见表 2-2。 表 2-2 促进宫颈癌细胞凋亡的作用 （J ±s)
细胞生长抑制率
药物浓度（ g/ml)
rSIFN-co 干复津
21.3 ±3.9 21.3 ±3.9
53.5 ±4.6①② 47.6±3.1①
64.9 ±3.3① 67.1 ±3.6①
〇 Ο 74.4 ± 1.3① 72.0 ± 2.6①
①与对照比较， ><0.01；
②与同浓度干复津组比较， 尸<0.01。 实施例 5 rSIFN-co与千复津的人体药动学及生物等效性研究 本实施例为重组干扰素 rSIFN-co及干复津人体药动学及生物等效性的研究。 分别以本发明所述重组干扰素 rSIFN-co和美国安进公司生产的干复津为试验药 物， 对其人体药动学和生物等效性进行比较研究。
干扰素在健康人体内的药物代谢动力学研究是干扰素研究的难点。由于药用 干扰素注射后在血浆中的干扰素水平均系微量， 用酶联免疫吸附测定（ELISA) 或病毒细胞病变抑制法都难以直接在健康成人血清中将其测出。 目前， 干扰素的 药物代谢动力学研究所采用的检测指标一般是 2'，5'-OAS ( 2-5A寡聚核苷酸酶）, 它既是干扰素的诱生产物， 又是其药效活力的代表。
一、 对象与方法
1.对象
纳入健康男性志愿者 18名， 年龄（22.8±1.4) 岁， 身高 （170±5.0) cm, 体 重指数 20·5±2.4 [体重（ 59.4±7.2 ) kg]。 受试者经全面体格检查、 实验室检查（包 括血液学、 尿常规、 肝肾功） 及心电图检查均正常。 自受试前 4周起未使用过 任何药物； 试验前 3个月内未用过已知对脏器有损害的药物； 对试验药物无过敏 史； 自愿受试并签署知情同意书。
2. 方法
本试验获得四川大学华西医院医学伦理委员会批准， 根据中华人民共和国 GCP相关指导原则进行。 2.1 材料
试验制剂：
冻干重组高效复合干扰素粉针剂 （实验制剂， 即本明所述重组干扰素 rSIFN-co, 9μ§/支）。 参比制剂： 干复津注射液（参比制剂， 9 g/支）， 美国安进 公司生产。
2-5A试剂盒：
Eiken' Radioimmunoassay试剂盒由日本荣研化学株式会社提供， 试剂盒含： ( 1 ) 1125-标记 2',5'-OAS; ( 2 )抗 2，，5，-OAS血清； ( 3 ) 2', 5 '-OAS标准瓶 (每瓶 含 0、 10、 30、 90、 270或 810 pmol/dL的 2，，5'-OAS; ( 4 )緩冲液； （5 ) 空白血 清； （ 6 ) 聚（ I ) 聚（ C )琼脂； （ 7 ) ATP; ( 8 )疏基乙醇； （ 9 ) 随行质控血清。
2.2 试验设计与给药方法
采用随机自身交叉对照试验设计， 18名受试者随机均分为 A、 B 两组， 每 组 9人， 分别于两个周期内交叉皮下注射试验制剂 rSIFN-co 9 g和干复津 9 g， 清洗期 1周。
每次试验前日晚 8时后禁食至次日晨用药后 2h, 试验当日晨 7:00于上臂三 角肌部作皮下注射。 试验当日统一用标准餐（不含高脂肪食物）， 禁止吸烟、 饮 酒及饮茶、 咖啡类饮料， 试验期间禁用一切药物， 禁止剧烈活动。
2.3 血样采集与检测
给药前抽取血样 4ml， 给药后 2、 6、 12、 18、 22、 24、 26、 30、 34、 38、 42 及 48h于对侧肘静脉取血约 3.5 ml, 立即离心分离血清置于 -20°C保存直至检测。
检测各血样中血清 2',5'-OAS浓度。
3 统计学方法
使用 DAS verl.O统计软件， 试验制剂和参比制剂的比较采用配对 t检验。 二、 结果
根据测得的各血样中血清 2，,5，-OAS浓度，绘制均值酶浓度 -时间曲线，见图
16。
由图 16可以看出， 分别皮下注射 rSIFN-co 9 g、 干复津 9 g后， 二者的酶 浓度-时间曲线变化趋势基本一致，但皮下注射 rSIFN- co血中酶浓度-时间曲线峰 浓度显著高于干复津。 试验制剂 （rSIFN-co)对参比制剂 （干复津） 的相对生物利用度（F)按下 式进行计算：
F=[AUC JAUC ]x [参比制剂剂量 /试验制剂剂量] χ100%
计算结果显示 rSIFN-co相对生物利用度 (F0-48)为 125.4%。 试验制剂和 参比制剂的 Tmax差异无统计学意义（t=1.458, P=0.163 ) , AUQ^和 Cmax差异 有统计学意义（t=2.730, P=0.014; t=2.347, P=0.031 ) , 试-睑制剂高于参比制剂。
此外， 比较两组的不良反应： 干复津组的不良反应在发生率、发生程度和持 续时间三个方面均重于 rSIFN-co组。
三、 结论
( 1 ) 注射 rSIFN-co与干复津后均能诱生 2'5 '-OAS, 二者的药代动力学曲 线变化趋势一致， 主要药代动力学参数比较无统计学差异。
( 2 ) 注射 rSIFN-co诱生的 2'5-OAS的 Cmax和 AUCo_48大于干复津诱生 的 2 5- OAS的 Cmax和 AUCM8, 提示 rSIFN-co在相同剂量治疗时， 其疗效可 能优于干复津。
(3)干复津组的不良反应在发生率、 发生程度和持续时间三个方面均重于 rSIFN-co组。
(4)以不同采血时间测定的受试者血清中 2,5"寡腺苷酸合成^(2,5-0八5 ) 的含量作图（即均值酶浓度 -时间曲线）后发现： 注射 rSIFN-co诱生的 2'5'-OAS 浓度一般出现双峰现象，其曲线下面积显著大于在同一条件下注射干复津所获得 的曲线下面积， 且并未由于曲线下面积的增加而增加不良反应发生率和 /或加重 发生程度。 实施例 6 重组干扰素的结晶
制备优质 rSIFN-co蛋白质单晶是测定其晶体结构的前提条件。 用于晶体生 长的 rSIFN-co样品得自如上所述的本发明 rSIFN-co rSIFN-co的单晶制备的方 法、 技术流程和结晶奈件， 及其晶体学参数如下：
将上述本发明的 rSIFN-co冻干粉溶解于纯水中并在低温 （ -20°C ) 下保存， 初始蛋白质浓度为 0.42mg/ml。结晶前将 rSIFN-co蛋白质样品浓缩至 3-3.5mg/ml 后立即用于晶体生长实验。 晶体培养均在室温下（293k )釆用悬滴汽相扩散法进 行。
在结晶研究初期， rSIFN-co的微晶可以在多个系列条件中出现，但获得可用 于 X-射线衍射分析并有足够分辨率的优质单晶十分困难。 经过大量结晶条件优 化研究， 得出所获晶体质量最好的结晶溶液条件为 1.2M ( mol/L，下同） Li2S04、 0.1M CAPS ( 3- (环己氨基） -1-丙磺酸）， H 11.1 , 0.02 M MgCl2。 按此条件配 制的结晶溶液静置三天至一周后， 获得 rSIFN-co蛋白质的优良单晶， 晶体 为三方晶型， 晶体大小为 0.42 X 0.08 X 0.08 mm。 用于 X-射线^ "射晶体结 构分析的 rSIFN-co蛋白质晶体见图 1。 实施例 7 晶体 X-射线衍射数据的分析
晶体衍射数据收集：
数据收集在日本筑波光子工厂同步辐射站线 BL5A, 低温条件下（ 100K )完 成。 具体操作步骤如下：
( 1 ) 在显微镜下仔细小心用安置晶体工具将晶体从母液捞到工具顶部的 线圈中；
( 2 ) 采用快速冷冻技术（Flash-Cooling )迅速将含有晶体的线圈在防 冻试剂 parrafin oil (购自 hampton Research公司）浸泡几秒后， 快速将安置晶体 工具放置在衍射仪器测角头上， 这时晶体也瞬间处于低温氮气流（ 100K ) 中， 使数据收集在 100K低温下进行；
( 3 ) 设定参数后开始进行数据收集， 光源波长为 1.0人， 探测器为 ADSC Quantum 315 CCD (电荷偶合器件探测器）， 晶体到探测器的距离 为 310mm; 采用回摆法收集数据， 每幅画面所用回摆角度为 1°, 每幅曝光 时间为 12秒， 总共收集了 110幅画面 （见图 2 )。
衍射数据处理和分析： 衍射实验首先收集得到一套直观的衍射画面（图 2), 这些画面必须经 过收集到的全套衍射数据用 CCP4程序包进行处理和分析， 以应用于衍射 数据质量评估和结构解析。 这个过程包括： 1) 指标化： 将衍射数据转换 为晶体学指标（h, k，l), 并计算出晶胞参数和空间群； 2)参数修正： 对晶 胞参数、 晶体和探测器间距离与角度和晶体镶嵌度等参数进行精化； 3) 积分： 从衍射斑点得到强度信息； 4) 数据合并： 将所有对称相关和重复 的衍射点合并， 产生一套由独立衍射点构成的完整数据； 5) 将强度数据 转化为结构振幅。 rSIFN-co晶体衍射数据收集和处理分析的结果详见表 3。 表 3
rSIFN-co晶体衍射数据收集和处理分析结果
数据采集条件
X射线源 PF,BL-5A
波长（A) 1.0
ADSC Quantum 检测器
CCD
距离 （mm) 310
温度（ K ) 100
数据采集统计
空间群（分子 不对称单位） P3】21 (2)
晶胞参数
a=b (人） 77.920,
c (A) 125.935
α=β=90°, γ=120°
溶剂含量（％) 56.7
分辨率范围 （Α) 67.58-2.60
衍射点 （ Ι/σ ( I ) >0 ) 86556
独特衍射点 （ Ι/σ ( I )〉0 ) 14052
最外壳层 (Α) 2.74-2.60
对称相关 ί射点质量因子 R ( % )： 全部，（最外壳层） 7.1 (25.8)
信噪比： 21.1 (4.5)
完整度 ( ) ：全部，（最外壳层） 99.5 ( 100.0 )
冗余度:全部，（最外壳层） 6.2 (6.5) 实施例 8 晶体结构解析
晶体衍射相位的测定和 rSIFN-co分子初始结构模型的建立
rSIFN-co 晶体结构采用分子置换法进行相位解析， 选择綿羊 IFN-τ (其与 rSIFN-co序列同源性 54% )的晶体结构（ PDB编号 1B5L )作为分子置换的同源 结构模型， 用软件程序 phaser, 进行旋转函数和平移函数的计算分析， 推定 rSIFN-co分子在晶胞中的位置和取向。根据劳厄群和系统消光规律， 测定空间群 为 P3121 ,同时将模型分子进行一些修改（即保留了 1B5L结构中的 13-25, 37-69, 79-101 , 114-151位残基），计算结果是： Z-score为 15.71 , IL-gain为 307.79 Clash 为 0。 晶胞中分子堆积合理， 且在分子置换过程中 IL-gain逐步上升， 表明获得 了正确解， 测定了各衍射点的初始相位。 进而用 Phaser生成的带有初始相位的 mtz通过 FFT生成电子密度图， 获得的 rSIFN-co分子的初始结构模型与生成的 电子密度图匹配良好， 表明已获得 rSIFN-co所有衍射点的正确相位解。 以所述 结果为基 ， 构建了 rSIFN-co分子的初始结构模型。 rSIFN-co结构模型的精化
运用分子模建技术和计算机优化程序， 对 rSIFN-co分子初始模型中的所有 非氢原子的坐标参数和温度因子进行叠代式修正 （refinement ), 以获得精确的 rSIFN-co分子结构模型。
结构修正采用程序 CNS 1.1 , 数据为没有相位的母体数据， 从其中随机提取 10 %作为测试集（testing set ), 且至始至终保持同一套随机选取的测试集。 结构 模型中的所有原子均参与修正， 且每个原子的修正参数均为 4个， 包括坐标（ X , y, z )和各向同性温度因子 B。 整个修正过程中， 结构的计算机自动修正与模型 的人工调整、 搭建（用软件 0 ) 交替进行， 在修正起始时使用了限制性的 NCS, 当在结构调整基本完成时， 修正中不再使用 NCS。 当 R k因子（低于 0.30 )和 Rfree因子基本不再继续下降时， 开始在结构中加入水和溶剂分子， 最终完成结构 的精化，其主要精化指标为， Rw。rk为 0.250, Rfree为 0.286。最终获得的精化 rSIFN-co 分子结构的主要指标列于表 4。 所得到的 rSIFN-co的原子坐标如表 7所示。 表 4
rSIFN-co分子精化结构模型的主要参数指标和质量统计结果 分辨率范围 （最外壳层）（人） 20.0-2.6 信噪比截止点 0.0 晶体学不符合指数（最外壳层 ) ( % ) 25.0 ( 36.3 ) 自由不符合指数（最外壳层） 、％、 28.6 ( 40.5 ) 不对称单位中的组分
所有的残基数 293
A链的残基数（未构建的残基数） 146 ( 20 )
B链的残基数（未构建的残基数） 147 ( 19 ) 水和溶剂分子数 123
均方根偏差
键长 （人 ) 0.007 键角 （° ) 1.379 二面角 （。） 19.234 不合适角度 （° ) 0.844 威尔逊温度因子 （人 2 ) 70.7 平均温度因子 （人 2 )
总的原子数 （2403 ) 61.76 蛋白质的原子数 （2254 ) 61.11 蛋白质 A链 ( 1120 ) 58.39 蛋白质 B链 ( 1134 ) 63.79 水和溶剂 （ 149 ) 68.21 拉氏构象图统计 （％) 3
最适区 90.6 附加允许区 9.1 一般允许区 0.4
不允许区 0.0
1 自由不符合指数 Rfree取不参与修正的 10%总衍射点数计算；
2相对标准键长 /键角计算的均方根偏差
3 拉氏构象图 （ Ramachandran plot )统计所用软件为 PROCHECK. 实施例 9 rSIFN-co分子结构模型的质量表征
rSIFN-co分子结构模型的质量表征
( 1 ) 模型的电子密度图： 可以直观、 清晰、 准确地显示 rSIFN-co。 图 3 为一代表性电子密度图与 rSIFN-co分子中氨基酸残基结构的配合情况， 可清晰 辨识不同氨基酸残基的空间位置和取向。
( 2 ) 平均温度因子随残基的分布图 （见图 4 )。
( 3 ) 分子的立体化学指标——拉氏构象图： rSIFN-co分子的立体化学合理 性由拉氏构象图 （图 5 ) 表征， 其结构位于最适允许区的氨基酸残基数占 90.6% , 位于允许区的 9.1% , 位于一般允许区的 0.4%。 表明 rSIFN-co分 子结构模型具有合理的立体化学。 实施例 10 rSIFN-co分子的晶体结构特征
rSIFN-co分子在晶体中的堆积及总体排列
rSIFN-co分子在晶胞中的堆积方式见图 6所示。 rSIFN-co晶体结构的一个不 对称单位中包含 2个蛋白质分子（称为晶体学二聚体）（见图 7所示）。 二聚体间 的包埋面积为 1033.3人 2 , 即每个晶体所贡献的面积为 516.6人2 , 仅占单体总面 积的约 6.4%。 二聚体中 A链的 A、 B、 F面与 B链的 C、 D、 E面相对应 （见图 9所示）； 用 VADAR软件计算单体，二聚体折叠自由能量分别为 -126.9和 -257.1 , 即二聚体折叠自由能与两个孤立单体自由能分别 （ -126.9x2 )很接近。 这表明二 聚体间相互作用里较弱。 同时两者间仅存在两个弱氢键 A12 ( ARG ) NH2...NH2 B71 ( Arg )， 3.05埃； A145 ( Arg ) NH1...OH B90 ( Tyr ) ， 3.14埃。
在纯化过程中证明 rSIFN-co蛋白质溶液态下为单体， 现有生化功能实验表 明 IFN-α类功能单位应是单体， 因此这个二聚体可能是由于晶体堆积而形成的。 二聚体结构
一个不对称单位中的两个 rSIFN-co单分子形成了一个二聚体。 图 8显示出 了 rSIFN-co 晶体学二聚体的组织方式。 A链由 11-103 和 111-163位残基构成 ( 1-10, 104- 110和 164-166由于在电子密度图上没有表现而没有构建）； B链由 11-103和 110-163位残基构成（ 1-10, 104-109和 164-166由于在电子密度图上没 有表现而没有构建）。在晶体结构中可见到每个单体的第 29位和第 139位的 Cys 形成了分子内二石克键， 而第 1位和第 99位的 Cys间的分子内二石克键由于第 1位 Cys在晶体结构中未构建所以没有体现。 另外， A链的 30-33、 47-49位及 B链 的 30-33、 48-50位残基由于侧链的密度没有表现， 故这些残基主要构建为 Ala 或 Gly。 另外， A链的 30-33、 47-49位及 B链的 30-33'、 48-50位残基由于侧链 的密度没有表现，故这些残基主要构建为 Ala或 Gly。两个单体的结构基本相同， 由非晶体学对称性所联系 （从 B到 A, 极角 Omega、 Phi, Kappa分别为 170.64、 94.56、 118.35; 平移分数 tx、 ty、 tz分别为 -1.061、 -0.225、 0.155 )。 对两个单体 进行叠合比较（见图 8 )， 除了少数几段分子表面的环（loop )区域柔性比较大以 外， 绝大部分残基能够很好地叠合（所有 α碳原子 RMSD随氨基酸的分布如图 8c ), 其中 127个残基（ 13-30、 34-44、 53-101、 115-163 ) α碳原子的均方根偏差 为 0.64埃。 局部结构的差异是可能由于蛋白质的较大柔性以及晶体堆积环境的 不同而造成的。 单分子结构
每个单体由 6段 α螺旋（A、 C、 C，、 D、 E、 F )、 一段 310螺旋（B ) 以及 它们之间的连接肽段所构成， 整个单体结构的折叠方式属于螺旋状细胞因子型 (见图 9所示）。 6段 α螺旋（A、 C、 C'、 D、 E、 F ) 所对应的氨基酸残基位置 分别是： 13-20、 50-68、 70-76、 79-100、 114-133、 138-160。 一段 310螺旋（B ) 所对应的氨基酸残基位置是 40-43。 图 9清楚地显示了这些二级结构的分布和组 织方式。 图 10示出了二级结构与氨基酸序列的对应关系。 实施例 11 rSIFN-co和 IFN-a2b 的三维结构
干扰素根据受体的不同，首先可分为两类： I型和 II型。 I型类群又可分为 a、 β、 ω等， 其中 IFNa又包括近 15种不同的亚型， 不同 a类蛋白质间的蛋白质序 列同源性都在 80%以上， 但是它们的功能却显示了多样性。 rSIFN- co蛋白质属 于 IFNa类中非天然的人工设计蛋白质，至今 I型 IFNs只有 6个三维结构（表 5 ), 它们的氨基酸序列同源性可见序列对比 （如图 11所示）。
从表 5 和图 11 对比分析可以知道， 与 rSIFN-co 的晶体结构最相近的是 IFN-a2b (如图 12所示）， 在序列上， rSIFN-co主要在 45位比 IFN-a2b多一个天 冬氨酸（ D ); 在三维结构上， 它们主要区别在 AB环 （ 25-33位残基）和 BC环
( 44-52位残基）的空间构象有显著不同。 IFN-a2b的晶体结构已在 2.9埃分辨率 解析， 但在蛋白质数据库 （ Protein Data Bank, PDB ) 中仅有 α碳原子的坐标， 所有其它原子的坐标数缺失（ PDB Code 1RH2 )。 故 rSIFN-co与 IFN-a2b的结构 比较只能在 α碳原子的水平上进行。这两个分子所有 α碳原子之间的均方根偏差
( RMSD ) 为 1.577埃， 但它们在 ΑΒ环区和 BC环区的均方根偏差为 3.6埃和 2.9埃， 分别是其总平均值的 2.5倍和 2倍。 此外， rSIFN-co的晶体结构中不对 称单位中含有 2个分子， 而 IFN-a2b的晶体学不对称单位中有 6个蛋白质分子， 由 3个二聚体构成。 显然， rSIFN-co的二聚体组织方式与 IFN-a2b明显不同（如 图 13所示）。 表 5
已测定结构的千扰素分子
蛋白质名称 来源 测定方法 分 辨 ^ PDB 编码 与 rSIFN-co的
( A ) 同一性 rSIFN-co 合成 X-ray 2.6 本发明
IFN-α 2b 人 X-ray 2.9 1RH2 (仅有 α 89% 碳原子编码）
IFN-a 2a 人 NMR 1ITF 88%
IFN-τ 人 X-ray 2.1 1B5L 54%
IFN-β 人 X-ray 2.2 1AU1 30%
IFN-β 鼠 X-ray 2.2 1RMI 23%
现有技术已知，作为细胞因子的干扰素首先与细胞膜上的特异受体结合， 以 激活多种信号传导途径， 使机体产生抗病毒、 抗肿瘤等生物学效应。 rSIFN-co 属于 IFN-α类干扰素，其在细胞膜上的受体已知由 IFNAR1和 IFNAR2共同组成， 并由此构建了受体与 IFN-a分子结合的三维结构模型 （如图 15a所示）。 以此为 基础，一系列分子生物学实验研究显示，IFN- α类蛋白盾与受体 IFNAR1、IFNAR2 相互作用时形成夹层式结构（如图 15a所示），即 IFN-α类的 A、B、F面与 IFNAR2 相互作用， 而其相对的 C、 D、 E面与 IFNAR1相作用。 同时， 深入的分子生物 学定位突变试 ¾揭示 IFN-a类功能重要区（如图 15所示），其中 AB环尤其重要， 它是与 IFNAR2相互作用的主要分子部位。结构比较显示（如图 12、表 6所示）， 这一重要部位在 rSIFN-co和 IFN-a2b中明显不同。 这一活性重要部位的结构差 异， 可能通过影响其与受体结合特征的改变， 产生不同的生理 /药理效应。
显然， rSIFN-co干扰素虽然与 IFN-a2b有类似的分子骨架，但却在其功能活 性重要区具有显著不同的结构。 因此， 从药理活性密切相关的分子局部结构看， rSIFN-co是不同于 IFN-a2b的新类型分子，从而导致了两者间具有显著不同的生 物学和药物学特征。 以特殊的关键部位的特征三维结构为基础， 干扰素 rSIFN-co 可能产生独特的生理和药理效应。 表 6
rSIFN-co与 IFN-a2b 的 AB环和 BC环中碳原子的位置均方根偏差（单位:
埃）
Figure imgf000047_0001
表 7
rSIFN-co的原子坐标
CRYST1 77. 920 77. 920 125. 935 90. 00 90. 00 120. 00 P 31 2 1
ATOM 1 CB ASN A 11 - 36. 673 14 399 -31. 951 1 00 79. 36 A
ATOM 2 CG ASN A 11 -37. 660 14. 647 -33. 090 1 00 81. 91 A
ATOM 3 0D1 ASN A 11 - 37. 274 14 829 -34. 245 1 00 85. 24 A
ATOM 4 ND2 ASN A 11 -38. 947 14 622 -32. 764 1 00 82. 54 A
ATOM 5 C ASN A 11 -34. 980 16 273 -31. 802 1 00 76. 68 A
ATOM 6 0 ASN A 11 -34. 061 16. 507 -31. 007 1 00 76. 57 A
ATOM 7 N ASN A 11 -34. 283 13. 985 -31. 533 1 00 78. 32 A
ATOM 8 CA ASN A 11 - 35. 239 14. 843 -32. 283 1 00 77. 86 A
ATOM 9 N ARG A 12 -35. 760 17. 226 -32. 307 1 00 74. 41 A
ATOM 10 CA ARG A 12 -35· 635 18. 622 -31. 899 1 00 69. 90 A
ATOM 11 CB ARG A 12 -35. 404 19. 525 -33. 115 1 00 72. 01 A
ATOM 12 CG ARG A 12 -34. 052 19. 300 -33. 792 1 00 77. 29 A
ATOM 13 CD ARG A 12 -33. 757 20. 318 -34. 894 1 00 79. 77 A
ATOM 14 NE ARG A 12 —32. 967 21. 461 -34. 430 1 00 83. 05 A V 00•I 69ε•LZ- L Z 'Zf- ΐ V nai 3 83 V
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ATOM 543 CD1 PHE A 85 -39. 076 22. 847 -23. 982 1. 00 43. 39 A
ATOM 544 CD2 PHE A 85 -39. 084 23. 734 -21. 768 1. 00 42. 83 A
ATOM 545 CE1 PHE A 85 -37. 897 23. 528 -24. 265 1. 00 42. 40 A
ATOM 546 CE2 PHE A 85 -37, 904 24. 417 -22. 043 1. 00 43. 37 A
ATOM 547 CZ PHE A 85 -37. 312 24. 313 - 23. 295 1. 00 42. 70 A
ATOM 548 C PHE A 85 -39. 813 20. 206 - 21. 572 1. 00 44. 30 A
ATOM 549 0 PHE A 85 -38. 835 19. 562 -21. 964 1. 00 45. 07 A
ATOM 550 N TYR A 86 -40. 014 20. 482 -20. 282 1. 00 43. 21 A
ATOM 551 CA TYR A 86 - 39. 018 20. 055 -19. 319 1. 00 44. 18 A
ATOM 552 CB TYR A 86 -39. 208 20. 748 -17. 948 1. 00 45. 30 A
ATOM 553 CG TYR A 86 -40. 455 20. 448 -17. 144 1. 00 44. 10 A
ATOM 554 CD1 TYR A 86 -41. 328 21. 474 -16. 797 1. 00 43. 43 A
ATOM 555 CE1 TYR A 86 - 42. 432 21. 239 -15. 988 1. 00 45. 72 A
ATOM 556 CD2 TYR A 86 -40. 720 19. 163 -16. 662 1. 00 43. 84 A
ATOM 557 CE2 TYR A 86 -41. 828 18. 915 -15. 846 1. 00 46. 21 A
ATOM 558 CZ TYR A 86 -42. 678 19. 963 -15, 513 1. 00 47. 37 A
ATOM 559 OH TYR A 86 - 43. 764 19. 756 -14. 691 1. 00 49. 38 A
ATOM 560 C TYR A 86 -38. 862 18. 549 -19. 164 1. 00 44. 37 A
ATOM 561 0 TYR A 86 -37. 848 18. 080 -18. 656 1. 00 44. 46 A
ATOM 562 N THR A 87 -39. 846 17. 785 -19. 621 1. 00 44. 44 A
ATOM 563 CA THR A 87 -39. 752 16. 330 -19. 537 1. 00 43. 78 A
ATOM 564 CB THR A 87 - 41. 129 15. 644 -19. 751 1. 00 43. 93 A
ATOM 565 0G1 THR A 87 -42. 035 16. 055 -18. 722 1. 00 42. 91 A
ATOM 566 CG2 THR A 87 -40. 986 14. 130 -19. 712 1. 00 40. 48 A
ATOM 567 C THR A 87 -38. 813 15. 905 -20. 654 1. 00 43. 41 A
ATOM 568 0 THR A 87 - 38. 040 14. 962 -20. 509 1. 00 42. 95 A
ATOM 569 N GLU A 88 -38. 898 16. 620 - 21. 774 1. 00 43. 70 A
ATOM 570 CA GLU A 88 -38. 057 16. 359 -22. 932 1. 00 42. 33 A
ATOM 571 CB GLU A 88 -38. 503 17. 245 -24. 098 1. 00 43. 68 A
ATOM 572 CG GLU A 88 -37. 754 17. 014 -25. 394 1. 00 48. 37 A
ATOM 573 CD GLU A 88 -37. 718 15. 546 -25. 822 1. 00 51. 49 A
ATOM 574 0E1 GLU A 88 -38. 767 14. 864 -25. 751 1. 00 51. 50 A
ATOM 575 0E2 GLU A 88 -36. 634 15. 083 -26. 242 1. 00 52. 60 A
ATOM 576 C GLU A 88 -36. 616 16. 664 -22. 541 1. 00 40. 76 A
ATOM 577 0 GLU A 88 -35. 695 15. 921 -22. 878 1. 00 40. 05 A
ATOM 578 N LEU A 89 -36. 428 17. 756 -21. 809 1. 00 39. 65 A
ATOM 579 CA LEU A 89 - 35. 096 18. 127 -21. 373 1. 00 40. 05 A
ATOM 580 CB LEU A 89 -35. 128 19. 464 -20. 619 1. 00 39. 26 A
ATOM 581 CG LEU A 89 - 35. 580 20. 688 -21. 432 1. 00 39. 90 A
ATOM 582 CD1 LEU A 89 -35. 594 21. 916 -20. 546 1. 00 41. 45 A
ATOM 583 CD2 LEU A 89 -34. 647 20. 917 -22. 599 1. 00 37. 56 A
ATOM 584 C LEU A 89 -34. 555 17. 030 - 20. 481 1. 00 40. 50 A
ATOM 585 0 LEU A 89 -33. 394 16. 638 - 20. 598 1. 00 39. 83 A
ATOM 586 N TYR A 90 -35. 412 16. 520 —19. 598 1. 00 42. 46 A
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V 'Of 00 "ΐ 99Ϊ ■8ΐ- OZI '82 80 ΊΖ- fZl V HHd zao WL WOIV
V IS 'Zf 00 ■I 6S6 'Ζΐ- LOO '62 ΖΖ6 ■6Ζ- Ul V 3Hd mo S8Z WOIV
V 01 'Zf 00 •ΐ Z9f "Ζΐ- 606 '82 Ζΐ9 SZ- fZl V 3Hd 03 ZSL WOIV
V 69 •It 00 •ΐ LfZ ·9ΐ- 889 '6Ζ 881 SZ- fZl V HHd ao Ϊ8Ζ WOIV
V Zf 'Zf 00 •ΐ 0Z ·9ΐ- Ζ8Ϊ •ιε £66 'ίτ- fZl V HHd VD 08Z WOIV
V LI 'Of 00 9Z0 'Ζΐ- 6Zf ■ιε 99 fZl V HHd N 6ZZ 皿 V
V 08 'Of 00 "Τ 988 •il- f9f •εε 08ΐ 'ι - ZZl V MAI 0 8ZZ KOIV
V f5 'Of 00 •ΐ 8ΪΖ 899 fL£ ·9Ζ- ZZl V HAI 3 ILL 皿 V
V 99 'St' 00 •ΐ 6SZ τζ- Zf6 •ιε 901 -6Ζ- ZI V Ml HO 9LL WOIV
V 8 "St' 00 •ΐ T9£ 'ΖΖ- 6 •ιε 00 ΐ ■82- ZZl V Ml Z3 5LL WOIV
V Z6 00 •ΐ 9Z9 -\ζ- f9L •οε 808 •ιζ- £Zl V HAJ, fll WOIV
V 00 •ΐ 6SZ •02- 19L •οε 89Ζ '92- £Zl V H入丄 zao UL KOiV
V LI 00 •ΐ OZI 'ΖΖ- 960 •εε 9ZS 'ΙΖ- ZZl V Ηλΐ UD ILL MOIV
V 96 00 'Τ m -\ζ- 6Z0 •εε η ■92- ZZl V HAJ, IQD ILL 葡 IV
V 92 ■ f 00 •ΐ ■οζ- 6 •ΐε ζιο "9S-
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V 00 00 •ΐ n ■61- •τε εζ8 ■η- ZZl V a入 1 83 69Z WOIV
V £L 'If 00 •ΐ 6 Z ■8Τ- Z89 •ζε 696 -fz- zz\ V Ml VO 89 10J.V
V 98 00 •ΐ 9ZZ 'Ζΐ- 10Z ■2ε 9S0 -η- ZZl V N Z9Z WOIV
V 6Z 00 •ΐ l •ST- 6Ϊ8 •εε 9Ζ9 ■u- ZZl V SAT 0 99i M)丄 V
V fL 00 •ΐ ISO ·9ΐ- 3ΐ8 ■ζζ 0£6 ζ~ ZZl V SA1 0 99Z WOIV
V ιε -89 00 •ΐ SZ8 - Z66 •εε Ζ£2 'Ζΐ- ZZl V SAT ZN f9L WOIV
V Z6 'f 00 •ΐ 269 ■fl- f66 •εε ΖΖ8 '8Τ- ZZl V SAT 33 Z9L WOIV
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V S9 '29 00 6Ϊ-9 ·9ΐ- "9δ 669 •ζε- 1£1 V nm ZQ3 098 皿 V
V o ·½ 00 'ΐ SSZ "5ΐ- £98 8ΐε m V nm IQD 6Ϊ-8 K01V
V ■£S 00 ■ΐ ΖΖ6 -9ΐ- ' 6 ·9ε- m V Π3Ί 03 WOIV
V 89 '99 00 'I 9S9 -fl- ζεο •9S 06S ·9ε- m V 93 WOIV
V 89 "89 00 ■ΐ Ζΐ9 -η- 899 •ζε εεζ ·9ε- m V nai V3 WO丄 V
V £8 •8S 00 'ΐ SZ8 -η- 06 ■ζε 8 V nai N 皿 V
V 6Z '69 00 'ΐ • - η\ 'Of •9S- οετ V 0 W01V
V K "69 00 •I Ζ96 ■ - 99ΐ n οει V a人丄 3 HOiV
V S8 '89 00 •ΐ 0f •OS- 8ζε επ ·9ε- οετ V Ml HO ZfS W01V
V ZZ •8S 00 ■ΐ SI9 ·6ΐ- S08 fLZ οετ V Ml ZO WO丄 V
V 9Z ·Ζ9 00 •I ZLZ ·6ΐ- 8Lf Έε LZf ■ee- οετ V Ml Z2D OfS WOIV
V 90 "89 00 Ί LZZ ·8ΐ- Ϊ06 ·8ε f6 ■η- OCT V Ml ZQD 6C8 WOIV
V ZZ •89 00 •I 020 •61- 099 6LZ ■κ- οει V a入丄 133 8C8 冊丄 V
V 90 •89 00 'ΐ ^ΖΟ ·8ΐ- fL6 Of 6Zf •εε- οετ V ΗλΙ IQD Z28 V
V 'Ζ9 00 •I 9ΐΖ 'Ζΐ- L£9 ■εε- 021 V ΗλΧ 93 9£8 皿 V
V OS •8S 00 •ΐ ΐΟΖ ■91- £66 ·8ε £99 'ζε- οει V HAl 93 9S8 W01V
V 66 '89 00 'ΐ '9ΐ- Ζ9£ ·6ε €96 ζ- οετ V V3 8 V
V 00 •09 00 Ί ΐ9ε ' - L6 ·8ε ΐ8ΐ 'ΖΖ- OGT V N εεδ 皿 V
V TO ■Ζ9 00 'ΐ ΐβΐ- 'ΖΙ- 019 '6ε 89Z, 6ΖΙ V nm 0 V
V fZ 19 00 •ΐ ζεο •ετ- S69 ·8ε 89ΐ •ζε- 6ΖΙ V nai 3 W01V
V Π •99 00 ■ΐ •ΐΐ- ΖΖ9 89Z ·6Ζ- 621 V nai zao 0S8 WOIV
V Of ' 9 00 •ΐ Ζ9Ζ •Οΐ- 6ΐ8 ·8ε Z86 'ΙΖ- 6ΖΙ V nai IQD 6Z8 WO丄 V
V 10 ■19 00 'ΐ ZL6 ■Οΐ- 9S0 ·6ε 82^ ■6Ζ- 6ΖΙ V nai 03 8Z8 WOIV
V ζε Έ9 00 'ΐ 99ΐ •Ζΐ - 9 ΐ ·8ε S98 •62- 6ΖΙ V nai 83 LZS W01V
V 98 •09 00 'ΐ Ζ\Ζ 'ΖΙ- 8ΙΖ •ζε ΙΖΐ, •ιε- 6Ζΐ V nai VD 9Z8 皿 V
V οε •6S 00 τ f L •21- οζε '9ε S8£ •ιε- 6ΖΙ V Π3Ί N gs8 皿 V
V zo ■99 00 Ί 5ff •Ζΐ- 2ΖΙ ·9ε Ζ09 •εε- 8ΖΙ V mi 0 WOIV
V Ϊ8 "99 00 •ΐ US "ΖΤ- 6 9 L1 'ΖΖ- 8Ζΐ V mi 0 εζ8 MOIV
V 9ΐ "99 00 'ΐ ε^6 •01- Ϊ69 •εε Ζ91 •ζε- 82ΐ V mi ZOO ZZ8 WOIV
V 99 ■99 00 ■ΐ 8Τ9 'ΖΙ- 86 'ιε 8ΖΙ V mi 190 128 舰 V
V 8ΐ S 00 ,ΐ 89S ·ζ\- Ζ£ ■εε Z9L ■ΐε- 8Ζΐ V mi 90 0Z8 WOIV
V 90 •SS 00 'ΐ •ει- LLZ Zlf 8Ζΐ V 肌 VD 6Ϊ8 moxv
V ZO ■Ζ 00 •ΐ -η- fLZ 699 •ιε- 8ΖΙ V mi N 8ΐ8 M01V
V 68 ■OS 00 ,ΐ 619 •SI- Ϊ68 ■9ε Ζΐ6 •ζε - V an 0 Ζΐ8 WOIV
V οε 'TS 00 "Τ ΐ 9 '9ΐ- 680 266 •ΐε-
Figure imgf000066_0001
V 311 3 9ΐ8 WOIV
V Ζ Ό9 00 'ΐ S^6 ■6ΐ- 6Π ιζι ■ΐε - ιζ\ V 3Ή WO 918 WOIV
V Ζ9 ■< 00 'ΐ 06Ζ ·8ΐ- •εε Π9 •οε- LZI V HI I TOO 8 WOIV
V V an ZOO ST8 WOIV
V
Figure imgf000066_0002
V an 93 Ζ18 WOIV
V 9 •OS 00 'ΐ ηβ ·9ΐ- V 311 V3 Π8 WOIV
V Ζ8 19 00 'ΐ ½9 ·9ΐ-
Figure imgf000066_0003
ΐ8ΐ ■ ε 89Α ·6Ζ- V 311 N 0ΐ8 WOIV
V 9ΐ •29 00 'ΐ Zf9 'SI- 89 ΐ •ζε 'οε- 9SI V OMV 0 608 WOIV
V 66 •is 00 'ΐ Ϊ68 -9ΐ- OLZ ·9ε S9S ·6Ζ- 9ΖΙ V oav 3 808 WOIV
V οε •AS 00 ,ΐ '6Ι- 8U ' ε 0S6 ζ- 9Ζ\ V OHV zm Α08 WOIV
SS0Z00/0l0iN3/X3d .8^,0/lTOi OAV ATOM 851 C LEU A 131 -36. 740 38. 193 -13. 330 1. 00 59. 15 A
ATOM 852 0 LEU A 131 -37. 811 38. 807 -13. 304 1. 00 57. 39 A
ATOM 853 N THR A 132 -35. 966 38. 041 -12. 262 1. 00 60. 31 A
ATOM 854 CA THR A 132 -36. 342 38. 608 -10. 982 1. 00 61. 04 A
ATOM 855 CB THR A 132 - 35. 474 38. 034 -9. 826 1 . 00 61. 40 A
ATOM 856 0G1 THR A 132 -35. 587 38. 887 -8. 680 1. 00 64. 65 A
ATOM 857 CG2 THR A 132 -34. 026 37. 929 -10. 227 1. 00 59. 77 A
ATOM 858 C THR A 132 -36. 212 40. 125 -1 1. 044 1. 00 61. 41 A
ATOM 859 0 THR A 132 -37. 1 12 40. 853 - 10. 624 1. 00 61. 07 A
ATOM 860 N GLY A 133 -35. 102 40. 598 -1 1. 597 1. 00 62. 03 A
ATOM 861 CA GLY A 133 -34. 891 42. 029 -11. 705 1. 00 62. 46 A
ATOM 862 C GLY A 133 -35. 929 42. 684 - 12, 592 1. 00 62. 83 A
ATOM 863 0 GLY A 133 -36. 032 43. 905 -12. 634 1 , 00 63. 96 A
ATOM 864 N LYS A 134 -36. 708 41. 869 -13. 295 1. 00 62. 98 A
ATOM 865 CA LYS A 134 -37. 729 42. 375 -14. 205 1. 00 62. 76 A
ATOM 866 CB LYS A 134 -37. 523 41. 775 - 15. 595 1. 00 63. 31 A
ATOM 867 CG LYS A 134 -37. 830 42. 712 -16. 742 1. 00 63. 91 A
ATOM 868 CD LYS A 134 -36. 734 43. 744 - 16. 932 1. 00 64. 03 A
ATOM 869 CE LYS A 134 -37. 008 44. 589 -18. 172 1. 00 65. 71 A
ATOM 870 NZ LYS A 134 - 35. 986 45. 656 -18. 394 1. 00 66. 60 A
ATOM 871 C LYS A 134 -39. 122 42. 026 -13. 694 1 , 00 62. 52 A
ATOM 872 0 LYS A 134 -40. 118 42. 159 -14. 408 1. 00 61. 64 A
ATOM 873 N ALA A 135 -39. 175 41. 558 - 12. 454 1. 00 62. 91 A
ATOM 874 CA ALA A 135 -40. 433 41. 207 -1 1. 809 1. 00 62. 68 A
ATOM 875 CB ALA A 135 - 41. 307 42. 469 - 1 1. 671 1. 00 63. 48 A
ATOM 876 C ALA A 135 -41. 221 40. 096 -12. 501 1. 00 61. 65 A
ATOM 877 0 ALA A 135 -42. 444 40. 041 -12. 385 1. 00 61. 18 A
ATOM 878 N TYR A 136 -40. 525 39. 215 -13. 213 1. 00 60. 39 A
ATOM 879 CA TYR A 136 -41. 166 38. 091 - 13. 908 1. 00 60. 51 A
ATOM 880 CB TYR A 136 -41. 622 37. 024 -12. 899 1. 00 60. 08 A
ATOM 881 CG TYR A 136 -40. 547 36. 596 -1 1. 924 1. 00 62. 10 A
ATOM 882 CD1 TYR A 136 -40. 241 37. 378 -10. 807 1. 00 62. 66 A
ATOM 883 CE1 TYR A 136 -39. 227 37. 007 -9. 919 1. 00 63. 23 A
ATOM 884 CD2 TYR A 136 -39. 811 35. 424 -12, 131 1. 00 62. 49 A
ATOM 885 CE2 TYR A 136 -38. 792 35. 044 -11. 249 1. 00 64. 05 A
ATOM 886 CZ TYR A 136 -38. 507 35. 844 -10. 146 1. 00 63. 71 A
ATOM 887 OH TYR A 136 -37. 495 35. 495 -9. 280 1. 00 63. 34 A
ATOM 888 C TYR A 136 - 42. 359 38. 479 -14. 785 1. 00 60. 49 A
ATOM 889 0 TYR A 136 -43. 334 37. 731 -14. 882 1. 00 60. 06 A
ATOM 890 N SER A 137 - 42. 289 39. 641 -15. 425 1. 00 61. 06 A
ATOM 891 CA SER A 137 -43. 383 40. 088 -16. 284 1. 00 61. 14 A
ATOM 892 CB SER A 137 - 43. 205 41. 557 -16, 650 1. 00 60. 90 A
ATOM 893 OG SER A 137 -42. 133 41. 713 -17. 559 1. 00 63. 48 A
ATOM 894 C SER A 137 -43. 432 39. 257 —17. 563 1. 00 61. 21 A / /,83/-onoz/S/DiOTOSId sso
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V 08 '99 00 •ΐ 66S •ΐε- Ζ69 ~η 999 - z- £91 V V1V 0 ran WOIV
V 99 '99 00 •ΐ 090 •ΐε- 6ZL 0^0 •6Z_ SSI V V1V 3 WOIV
V 9Z ■£S 00 Ί 869 •ιε- 6 0 'ι ΖΟ ιε- C9T V V1V eo ZZQ\ WOIV
V 69 •SS 00 •ΐ ILL •οε- 096 ΙΖ •οε- SSI V V1V VD ΐΖΟΐ TOIV
V £9 "S9 00 •ΐ 99S ■6Ζ- 69S '92 2Ζ9 ■οε- ε9ΐ V V1V N οζοτ M01V
V 82 19 00 •ΐ Ζ19 SZ- 6TS ' Ζ 980 •οε- Z 1 V aHd 0 6ΐ0ΐ MUV
V 'Z 00 •ΐ Z8S Z6f '92 9 ε •οε- Z51 V 3 8ΐ0ΐ IOIV
V 8S 00 •ΐ 9ΖΖ -fz- 6f9 ΊΖ •ΐε- Z 1 V 3Hd Z3 Ζΐθΐ 隐 V
V S9 ■6f 00 •ΐ LW ■9Ζ- 8S6 '\Ζ OSf ■ζζ- Z 1 V d ΖΆ3 9ΐ0ΐ 冊丄 V
V ZO 00 ■I ΟΐΟ 99 'ττ 90f •ιε- Ζ9Ϊ V md ΙΆ3 STOT TO丄 V
V 69 00 "1 9S9 ■9Ζ- 9fZ 0£f ■ζζ- Ζ9Ϊ V 3Hd zao WOIV
V 60 00 •ΐ U • z- 0Z6 f Z ■ιε- Z 1 V HHd ι ο ετοΐ WO丄 V
V Z6 00 •ΐ ZLL OLZ - ζ ZL8 •τε- Z 1 V 3Hd 03 Ζΐθΐ WOIV
V 06 00 ■ΐ τοε ·9Ζ- Z99 808 •ΐε- Z51 V 3Hd 93 ΐΐθΐ WOIV
V ZL ■OS 00 ■ΐ 8S6 ■9Ζ- fZO •οε- Z 1 V 3Hd VD ΟΤΟΐ WOIV
V 90 ■6f 00 'ΐ S96 '9S- 89f •ιζ LIZ •οε- Z 1 V 3Hd N 600 ΐ WOIV
V 99 ■9f 00 •ΐ Ζΐ9 'ιζ- SLZ •LZ επ ·8Ζ- 191 V aas 0 800ΐ WOIV
V 09 •8 00 •ΐ £ Ζ ■LZ- 6 6 •LZ 060 '6Ζ- Ι9Ϊ V aas 3 ΟΟΐ 皿 V
V LZ ■f9 00 'ΐ ■SZ- 006 ■6Ζ 088 ■6Ζ- Ϊ9Ϊ V Has 90 9001 WOIV
V Z6 00 •ΐ ZS8 -9Ζ- C66 '62 flL SZ- ΐ9ΐ V Has 93 900ΐ H01V
V Z9 00 •ΐ S Z •LZ- Z6f ζ U6 SZ- ΐ9ΐ V V3 ，00 ΐ WOIV
V ZL 00 ■ΐ ΊΖ- LL\ •οε ZZl •οε- ΐ3ΐ V H3S N εοοτ WOIV
V 99 00 'ΐ '6Ζ- ZL Z19 •62- 09ΐ V 0 ΖΟΟΐ WOIV
V ZZ 00 'ΐ ΐ9ΐ -&ζ- 9ZI •οε •οε- 09ΐ V oav 3 ΐθθΐ WOIV
V εε •69 00 •ΐ 0ΐ9 S88 ·9ε 6ZI 'ίΖ- 091 V oav zm ΟΟΟΐ HOIV
V S6 ΌΖ 00 •ΐ 88 •οε- £6^ ■9ε ggo SZ- 091 V oav ΪΗΝ 666 moiv
V If '69 00 •ΐ εΐ9 ff9 - ε 9Z6 • - OST V oav ZD 866 MOIV
V 61 •99 00 •ΐ 9 ■ΐε- 09S ε fL 09Τ V oav 3N Ζ66 WOIV
V S8 •69 00 •ΐ 9ΐ9 ■οε- 661 L\L ■6Ζ- OST V oav CD 966 WOIV
V S6 •S9 00 •ΐ ZIL 'οε- ILL •ζε Τ6Ϊ •οε- 09Τ V oav 93 966 KOIV
V 6f 00 ■ΐ OZL ·6Ζ- 68C •ζε f Z 'ιε- 09ΐ V DHV 93 ½6 WOIV
V LI 00 •ΐ 60Ζ ·6Ζ- S68 •οε 0 •ιε - 09Τ V oav VD S66 WOIV
V Ϊ9 00 •ΐ 5^6 ·8Ζ- 629 •οε εεζ •ζε- 09ΐ V oav N 266 皿 V
V LZ ■9f 00 ■ΐ 9ΖΖ '6Ζ- ε^9 '82 fZ6 •ζε- βη V law 0 166 WOIV
V If ■ f 00 ■ΐ Ζ 0 '6Ζ- •62 ZfZ •εε- 6U V 通 3 066 TO丄 V
V 01 ■Z 00 •ΐ •οε- 9C0 'ιζ ■9ε- V law 33 686 WOIV
V Zf •£9 00 •ΐ ■οε- πε τεε ■ζε- βη V 通 as 886 WOIV
V SL 'If 00 •ΐ IfZ •οε- 099 9ε ΐ ·9ε- 6fl V 00 86 ffi V
V '9ΐ- 00 •ΐ on '82- zso •οε 89Z •ge- βη V im go 986 WOIV
V 99 00 •ΐ OIZ '82- ·6Ζ f69 •^ε- V i V3 986 WOIV
V 9ΐ 00 •ΐ 068 '92- 0Ζ2 '6Ζ L6Z •^ε- β V law N ½6 WOIV
V 96 'Of 00 •ΐ 019 •92- 68^ '82 IZ •ζε— V an 0 £86 WOIV
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Figure imgf000074_0002
g s νΊν— •
Figure imgf000075_0001
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ATOM 1247 CA GLY B 32 -65. 360 16. 951 -50. 609 1. , 00 94. 95 B
ATOM 1248 C GLY B 32 -64. 893 18. 350 - 50. 254 1. , 00 93. 64 B
ATOM 1249 0 GLY B 32 -64. 396 18. 597 -49. 150 1. , 00 93. 49 B
ATOM 1250 N GLY B 33 -65. 052 19. 265 - 51. 207 1. 00 92. 18 B
ATOM 1251 CA GLY B 33 -64. 646 20. 645 -51. 009 1. 00 89. 82 B
ATOM 1252 C GLY B 33 -65. 345 21. 318 -49. 846 1. 00 88. 28 B
ATOM 1253 0 GLY B 33 -66. 577 21. 331 -49. 762 1. 00 88. 34 B
ATOM 1254 N HIS B 34 -64. 544 21 , 878 - 48. 943 1. 00 85. 84 B
ATOM 1255 CA HIS B 34 -65. 053 22. 571 -47. 762 1. 00 82. 36 B
ATOM 1256 CB HIS B 34 -64. 630 21. 808 -46. 496 1. 00 80. 60 B
ATOM 1257 CG HIS B 34 -65. 146 22, 398 - 45. 220 1. 00 78. 05 B
ATOM 1258 CD2 HIS B 34 -65. 986 21. 899 -44. 281 1. 00 76. 62 B
ATOM 1259 ND1 HIS B 34 -64. 766 23. 644 -44. 763 1. 00 77. 24 B
ATOM 1260 CE1 HIS B 34 -65. 346 23. 883 -43. 603 1. 00 75. 05 B
ATOM 1261 NE2 HIS B 34 -66. 092 22. 838 -43. 287 1. 00 75. 00 B
ATOM 1262 C HIS B 34 -64. 472 23. 983 -47. 764 1. 00 80. 50 B
ATOM 1263 0 HIS B 34 - 63. 349 24. 198 -48. 226 1. 00 81. 27 B
ATOM 1264 N ASP B 35 -65. 246 24. 947 -47. 278 1. 00 77. 74 B
ATOM 1265 CA ASP B 35 -64. 787 26. 330 -47. 225 1. 00 75. 78 B
ATOM 1266 CB ASP B 35 -65. 795 27. 264 -47. 895 1. 00 76. 50 B
ATOM 1267 CG ASP B 35 -65. 703 28. 687 -47. 371 1. 00 77. 21 B
ATOM 1268 0D1 ASP B 35 -64. 578 29. 227 -47. 288 1. 00 77. 48 B
ATOM 1269 0D2 ASP B 35 -66. 759 29. 266 -47. 040 1. 00 77. 57 B
ATOM 1270 C ASP B 35 -64. 579 26. 767 -45. 784 1. 00 73. 69 B
ATOM 1271 0 ASP B 35 -65. 486 26. 653 -44. 956 1. 00 74. 03 B
ATOM 1272 N PHE B 36 -63. 390 27. 282 -45. 484 1. 00 69. 71 B
ATOM 1273 CA PHE B 36 -63. 097 27. 707 -44. 125 1. 00 65. 47 B
ATOM 1274 CB PHE B 36 - 61. 694 27. 260 -43. 724 1. 00 63. 29 B
ATOM 1275 CG PHE B 36 -61. 484 25. 780 -43. 842 1. 00 61. 40 B
ATOM 1276 CD1 PHE B 36 -61. 068 25. 218 -45. 040 1. 00 60. 82 B
ATOM 1277 CD2 PHE B 36 -61. 722 24. 942 -42. 762 1. 00 59. 30 B
ATOM 1278 CE1 PHE B 36 -60. 896 23. 840 -45. 157 1. 00 59. 96 B
ATOM 1279 CE2 PHE B 36 -61. 554 23. 568 -42. 873 1. 00 58. 76 B
ATOM 1280 CZ PHE B 36 -61. 139 23. 018 - 44. 071 1. 00 57. 07 B
ATOM 1281 C PHE B 36 -63. 254 29. 195 -43. 882 1. 00 63. 46 B
ATOM 1282 0 PHE B 36 -62. 813 29. 701 - 42. 860 1. 00 63. 36 B
ATOM 1283 N GLY B 37 -63. 892 29. 889 -44. 816 1. 00 61. 85 B
ATOM 1284 CA GLY B 37 -64. 105 31. 317 - 44. 657 1. 00 60. 22 B
ATOM 1285 C GLY B 37 -62. 860 32. 105 -44. 299 1. 00 59. 45 B
ATOM 1286 0 GLY B 37 -62. 897 32. 984 -43. 436 1. 00 59. 52 B
ATOM 1287 N PHE B 38 -61. 757 31. 785 -44. 965 1. 00 58. 21 B
ATOM 1288 CA PHE B 38 -60. 496 32. 467 -44. 735 1. 00 57. 09 B
ATOM 1289 CB PHE B 38 -59. 465 32. 035 -45. 776 1. 00 55. 16 B
ATOM 1290 CG PHE B 38 -58. 169 32. 774 -45. 684 1. 00 52. 52 B
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SSOiOO/OTOZN3/X3d L8 ZL0/U0Z OAV ATOM 1599 C SER B 80 -38. 818 16. 577 -43 280 1 00 50. 52 B
ATOM 1600 0 SER B 80 -38. 223 17. 649 -43. 113 1 00 51. 02 B
ATOM 1601 N LEU B 81 -39. 937 16. 487 -43. 990 1 00 47. 40 B
ATOM 1602 CA LEU B 81 -40. 521 17. 684 -44. 574 1 00 46. 68 B
ATOM 1603 CB LEU B 81 - 41. 665 17. 327 -45. 531 1 00 44. 63 B
ATOM 1604 CG LEU B 81 -41. 253 16. 605 -46. 824 1 00 43. 12 B
ATOM 1605 CD1 LEU B 81 -42. 433 16. 565 - 47. 792 1 00 40. 61 B
ATOM 1606 CD2 LEU B 81 -40. 080 17. 329 -47. 467 1 00 38. 50 B
ATOM 1607 C LEU B 81 -41. 021 18. 607 -43. 464 1 00 47. 01 B
ATOM 1608 0 LEU B 81 - 40. 806 19. 823 -43. 507 1 00 46. 43 B
ATOM 1609 N LEU B 82 -41. 668 18. 019 -42. 462 1 00 45. 79 B
ATOM 1610 CA LEU B 82 -42. 184 18. 790 -41. 344 1 00 44. 38 B
ATOM 1611 CB LEU B 82 -42. 915 17. 881 -40. 355 1. 00 43. 65 B
ATOM 1612 CG LEU B 82 -44. 350 17. 506 -40. 712 1 00 42. 39 B
ATOM 1613 CD1 LEU B 82 -44. 969 16. 779 -39. 542 1 00 41. 48 B
ATOM 1614 CD2 LEU B 82 -45. 148 18. 764 - 41. 040 1 00 40. 02 B
ATOM 1615 C LEU B 82 -41. 106 19. 579 -40. 608 1 00 43. 60 B
ATOM 1616 0 LEU B 82 -41. 306 20. 748 -40. 294 1 00 43. 44 B
ATOM 1617 N ALA B 83 -39. 968 18. 958 -40. 326 1 00 41. 84 B
ATOM 1618 CA ALA B 83 -38. 920 19. 686 -39. 617 1 00 42. 47 B
ATOM 1619 CB ALA B 83 -37. 732 18. 749 -39. 241 1 00 42. 87 B
ATOM 1620 C ALA B 83 -38. 442 20. 857 -40. 466 1 00 40. 93 B
ATOM 1621 0 ALA B 83 - 38. 074 21. 898 -39. 930 1 00 41. 51 B
ATOM 1622 N LYS B 84 -38. 458 20. 693 -41. 785 1. 00 39. 80 B
ATOM 1623 CA LYS B 84 - 38. 041 21. 770 -42. 680 1 00 40. 20 B
ATOM 1624 CB LYS B 84 -37. 916 21. 257 -44. 121 1 00 42. 21 B
ATOM 1625 CG LYS B 84 -36. 919 20. 117 -44. 326 1. 00 44. 38 B
ATOM 1626 CD LYS B 84 -36. 542 20. 019 -45. 799 1. 00 45. 90 B
ATOM 1627 CE LYS B 84 -35. 545 18. 920 -46. 068 1 00 45. 47 B
ATOM 1628 NZ LYS B 84 -36. 209 17. 606 -45. 922 1 00 49. 90 B
ATOM 1629 C LYS B 84 -39. 076 22. 902 -42. 631 1. 00 39. 06 B
ATOM 1630 0 LYS B 84 -38. 743 24. 087 -42. 678 1 00 39. 36 B
ATOM 1631 N PHE B 85 -40. 338 22. 512 -42. 539 1 00 37. 58 B
ATOM 1632 CA PHE B 85 -41. 449 23. 442 -42. 475 1. 00 38. 00 B
ATOM 1633 CB PHE B 85 - 42. 749 22. 644 -42. 492 1. 00 38. 99 B
ATOM 1634 CG PHE B 85 -43. 959 23. 458 -42. 811 1 00 39. 63 B
ATOM 1635 CD1 PHE B 85 -43. 942 24. 366 -43. 866 1 00 38. 44 B
ATOM 1636 CD2 PHE B 85 - 45. 137 23. 276 -42. 099 1. 00 38. 81 B
ATOM 1637 CE1 PHE B 85 -45. 076 25. 079 -44. 211 1. 00 39. 59 B
ATOM 1638 CE2 PHE B 85 - 46. 288 23. 986 -42. 439 1 00 41. 20 B
ATOM 1639 CZ PHE B 85 -46. 255 24. 891 -43. 501 1. 00 41. 17 B
ATOM 1640 C PHE B 85 -41. 387 24. 331 -41. 223 1. 00 38. 99 B
ATOM 1641 0 PHE B 85 -41. 419 25. 557 -41. 334 1. 00 39. 44 B
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Claims

权利要求书 、 一种包含 SEQ ID NO:l所示的氨基酸序列的重组干扰素的晶体。 、 权利要求 1的重组干扰素晶体， 其中该晶体的空间群为 Ρ3,21。 、 权利要求 1-2任一项的重组干扰素晶体， 其中单晶胞参数为： a=b=77.92 埃， c=125.935埃， α = β =90°, γ =120°, 所有晶胞参数的可变性不超过
5%。 、 权利要求 1-3任一项的重组干扰素晶体， 所述晶体在一个不对称单位中包 含 2个分子。 、 权利要求 1-4任一项的重组干扰素晶体，进一步包含共价或非共价结合的 金属离子。 、 权利要求 1-5任一项的重组干扰素晶体，其中所述重组干扰素由包含 SEQ ID NO: 2的核苷酸序列所编码。 、 一种包含权利要求 1-6任一项的重组干扰素晶体的组合物。 、 权利要求 7的组合物， 所述组合物是药物组合物。 、 权利要求 8的组合物， 进一步包含有药学上可接受的载体。 、 一种基于计算机的答定能与重组干扰素相互作用的候选化合物的方 法， 所述方法包括步骤：
(a) 提供包含表 7所示的重组干扰素的原子坐标的三维结构， 所述原子 坐标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保守 残基骨架原子优选 Ca原子的均方根偏差内变化； 和
(b) 选择具有能与上述三维结构或该三维结构经选择的部分相互作用的 结构特征的候选化合物，从而鉴定能与所述重组干扰素相互作用的候选化 合物。 、 权利要求 10的方法，其中所述结构特征选自抗原性位点、亲水特性、 表面可接近性和结构基序。 、 权利要求 10-11任一项的方法， 其中步骤（b ) 中的候选化合物的选 择和鉴定包括：
(i) 对多个候选化合物生成三维结构； 和
(ii) 将步骤（ i )的三维结构各自与上述步骤（ a )中的三维结构或该三维 结构经选择的部分进行拟合， 以发现在能量上最有利的相互作用， 从而鉴定能与所述重组千扰素相互作用的候选化合物。 、 权利要求 10-12任一项的方法， 进一步包括步骤：
(c)获取或合成候选化合物； 和
(d)使所述候选化合物与所述重组干扰素接触以确定该候选化合物与所 述重组干扰素相互作用的能力。 、 权利要求 13的方法， 其中所述确定候选化合物与重组干扰素相互作 用的能力的步骤进一步包括测定所述重组干扰素与候选化合物接触时的 活性。 、 权利要求 14的方法， 其中所述活性选自抗病毒活性、 抗肿瘤活性、 抗增殖活性、 天然杀伤细胞活性和免疫调节活性。 、 权利要求 10-15任一项的方法，其中所述候选化合物是与所述重组干 扰素或其经选择的部分结合的配体。 、 权利要求 16的方法， 其中所述配体选自： 受体、 改性剂、 激动剂和 拮抗剂， 以及其中所述经选择的部分包含来自所述重组干扰素的第 25-33 位氨基酸残基和 /或第 44-52位氨基酸残基的一个或更多个氨基酸残基。 、 权利要求 17的方法， 其中所述经选择的部分包含来自所述重组干扰 素的第 25-33位氨基酸残基和 /或第 44-52位氨基酸残基。 、 一种基于计算机设计重组干扰素模拟物的方法， 包括步骤：
(a)对多个模拟物生成三维结构； 和
(b)将步骤（ a )的多个模拟物的三维结构各自与包含表 7所示的重组干扰 素的原子坐标的三维结构或该三维结构经选择的部分进行拟合， 以发 现所述重组干扰素的最佳拟合模拟物， 所述原子坐标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Ca原子的均方根偏差内变化。 、 一种基于计算机的合理药物设计方法， 包括步骤：
(a)提供包含表 7所示的重组干扰素的原子坐标的三维结构， 所述原子坐 标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保守 残基骨架原子优选 Ca原子的均方根偏差内变化；
(b)提供多个分子片段， 并对其生成三维结构；
(c)将所述多个分子片段的三维结构各自与步骤（ a )的三维结构或该三维 结构经选择的部分进行拟合； 和
(d)将经选择的所述分子片段组装到一个分子中以产生候选药物。 、 权利要求 20的方法， 进一步包括步骤：
(e)获得或合成候选药物； 和
(f) 令所述候选药物与所述重组干扰素接触， 从而确定候选药物与所述重 组干扰素相互作用的能力。 、 权利要求 10-21任一项的方法，其中所述三维结构经选择的部分包含 表 7中所包含的来自第 25-33位氨基酸残基和 /或第 44-52位氨基酸残基的 一个或更多个氨基酸残基的原子坐标，所述原子坐标任选地在小于约 0.65 埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Ca原子 的均方根偏差内变化。 、 权利要求 22的方法， 其中所述三维结构经选择的部分包含表 7中所 包含的来自第 25-33位氨基酸残基和 /或第 44-52位氨基酸残基的原子坐 标， 所述原子坐标任选地在小于约 0.65埃， 优选约 0.5埃以及更优选约 0.35埃的保守残基骨架原子优选 Cot原子的均方根偏差内变化。 、 一种干扰素模拟物，其包含如 SEQ ID NO:4和 /或 SEQ ID NO:5所示 的氨基酸序列。 、 权利要求 24的干扰素模拟物， 其中将所述干扰素模拟物三维结构中 的 α碳原子骨架与 IFN-a2b 三维结构中的 a碳原子骨架以最小二乘法方 式进行叠合后， 该干扰素模拟物在对应于 IFNa2b蛋白质分子中第 25-33 位残基上的各 a碳原子与 IFNa2b蛋白质分子在对应位残基上的 a碳原子 均方根偏差为 3.63埃 ±5%。 、 权利要求 25的干扰素模拟物， 其中与 IFNa2b对应位残基相比， 所 述干扰素模拟物在第 25-33位残基上的 a碳原子偏差分别是 3.291埃 ±5%、 4.779埃 ±5%、 5.090埃 ±5%、 3.588埃 ±5%、 2.567埃 ±5%、 2.437埃 ±5<¾、 3.526埃 ±5%、 4.820埃 ±5%和 2.756埃 ±5%。 、 权利要求 24-26任一项的干扰素模拟物，其中将所述干扰素模拟物三 维结构中的 a碳原子骨架与 IFN-a2b 三维结构中的 a碳原子骨架以最小 二乘法方式进行叠合后， 该干扰素模拟物在对应于 IFNa2b蛋白质分子中 第 44-52位残基上的各 a碳原子与 IFNa2b蛋白质分子在对应位残基上的 a碳原子均方才艮偏差为 2.90埃 ±5%。 、 权利要求 27的千扰素模拟物， 其中与 IFNa2b对应位残基相比， 所 述干扰素模拟物在第 44-52位残基上的 a碳原子偏差分别是 1.614埃 ±5%、 1.383埃 ±5%、 2.735埃 ±5%、 2.709埃 ±5%、 5.018埃 ±5%、 4.140埃 ±5%、 3.809埃 ±5%、 2.970埃 ±5%和 0.881埃 ±5%。 、 权利要求 24-28任一项的干扰素模拟物，其是功能性模拟物或结构性 模拟物。 、 权利要求 24-29任一项的干扰素模拟物，其中所述干扰素是重组千扰 素 ( rSIFN-co )。 、 包含权利要求 24-30任一项的干扰素模拟物的组合物。 、 权利要求 31的组合物， 所述组合物是药物组合物。 、 权利要求 32的組合物， 进一步包含有药学上可接受的载体。 、 一种治疗病毒性疾病和 /或肿瘤的方法， 所述方法包括向受试者施用 有效量的权利要求 1-6任一项的重组干扰素晶体、 权利要求 24- 30任一项 的干扰素模拟物或权利要求 7-9和 31-33任一项的组合物。 、 权利要求 1-6任一项的重组干扰素晶体、权利要求 24-30任一项的干 扰素模拟物或权利要求 7-9和 31-33任一项的组合物在制备用于治疗病毒 性疾病和 /或肿瘤的药物中的应用。 、 权利要求 34的方法或权利要求 35的应用， 其中所述病毒性疾病包 括： 曱型肝炎、 乙型肝炎、 丙型肝炎、 其它类型的肝炎、 由 Epstein-Barr 病毒、 人体免疫缺陷病毒、 埃博拉病毒、 严重急性呼吸道综合征病毒
( SARS ) 、 流感病毒、 巨细胞病毒、 单纯疱疹病毒、 或其它类型的疱疹 病毒、 乳头多瘤空泡病毒、 痘病毒、 小核糖核酸病毒、 腺病毒、 鼻病毒、 I型人类 T细胞白血病病毒、 或者 II型人类 T细胞白血病病毒， 或者 III 型人类 T细胞白血病病毒引起的病毒感染。 、 权利要求 34的方法或权利要求 35的应用， 其中所述肿瘤是癌症。 、 权利要求 34的方法或权利要求 35的应用， 其中所述肿瘤是实体瘤。 、 权利要求 34的方法或权利要求 35的应用， 其中所述肿瘤包括： 皮 肤癌、基底细胞癌及恶性黑色素瘤、 肾细胞癌、肝癌、 曱状腺癌、鼻咽癌、 实体肿瘤、 前列腺癌、 胃 /腹癌、 食道癌、 直肠癌、 胰癌、 乳腺癌、 卵巢 癌、 浅表膀胱癌、 血管瘤、 表皮样癌、 子宫颈癌、 非小细胞肺癌、 小细胞 肺癌、 神经胶质瘤、 血癌、 急性血癌、 慢性血癌、 慢性骨髓性白血病、 毛 细胞白血病、 淋巴腺瘤、 多发性骨髓瘤、 红血球过多病、 卡波济氏肉瘤。
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WO2014106459A2 (en) 2013-01-07 2014-07-10 Superlab Far East Limited A method for treating tumor by using recombinant interferon with changed spatial configuration
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EP3895725A2 (en) 2013-01-07 2021-10-20 Superlab Far East Limited A method for treating tumor by using recombinant interferon with changed spatial configuration
JP2022031331A (ja) * 2013-01-07 2022-02-18 スーパーラブ ファー イースト リミテッド 空間配置が変化した組換えインターフェロンを用いる腫瘍治療の方法

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